In vitro nephrotoxicity screening assay

ABSTRACT

The invention relates to methods for predicting the in vivo nephrotoxicity of a nucleic acid molecule, in particular a nucleic acid molecule such as a siRNA or an antisense oligonucleotide using an in vitro cell based assay measuring the levels of EGFR as toxicity biomarker, potentially in combination with other biomarkers like ATP and KIM-1.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application and claims priority under35 U.S.C. § 120 of application Ser. No. 16/310,505 filed Dec. 17, c2018,which is a National Phase application under 35 U.S.C. § 371 of PCTInternational Application number PCT/EP2017/064730 filed Jun. 16, 2017,which claims priority to EP Patent Application No. 16174994.0 filed Jun.17, 2016. The entire contents of the foregoing applications are herebyincorporated by reference.

SEQUENCE LISTING

This application contains a Sequence Listing that has been submittedelectronically as an XML file named “22460-0096002_SL_ST26.XML.” The XMLfile, created on May 15, 2023, is 18,898 bytes in size. The material inthe XML file is hereby incorporated by reference in its entirety.

FIELD OF INVENTION

The invention relates to methods for predicting the in vivonephrotoxicity of a nucleic acid molecule, in particular a nucleic acidmolecule such as a siRNA, an antisense oligonucleotide or aptamer usingan in vitro cell based assay measuring the levels of epidermal growthfactor receptor (EGFR) as toxicity biomarker, potentially in combinationwith other biomarkers like kidney injury molecule-1 (KIM-1) andadenosine triphosphate (ATP). The invention further relates to methodsfor selecting one or more nucleic acid molecules for in vivoadministration from a library of nucleic acid molecules, in particular anucleic acid molecule such as a RNAi agent, an antisense oligonucleotideor aptamer, using said assay.

BACKGROUND

One of the issues in identification of new as well as optimized drugcandidates is the event of dose limiting toxicity. When evaluated invivo, typically a sub-set of nucleic acid molecule compounds will elicita toxicity phenotype, such as drug induced kidney injury ornephrotoxicity.

In the past few years a number of models for predicting nephrotoxicityof nucleic acid molecules have been developed using various biomarkers.

Sohn et al 2013 Toxicology letters Vol 217 pp235 and Huang et al 2015Pharmacological Research & Perspectives Vol 3 pp e00148 both disclose invitro cell based assays for the prediction of nephrotoxicity of smallmolecules using various biomarkers including kidney injury molecule-1(KIM-1).

Ju et al 2015 Science translational medicine Vol 7, pp316ra193 describesepidermal growth factor (EGF) as a potential in vivo biomarker forchronic kidney disease by measuring EGF transcript and secretion in theurine correlated to the glomerular filtration rate. There is nodescription of measuring in vitro EGFR reduction or extracellular EGFuptake.

Wilmer et al 2016 Trends in Biotechnology Vol 34 pp 156 reviews furtherbiomarkers for screening of drug-induced nephrotoxicity both in vivo andin vitro (see table 2).

In vitro nephrotoxicity assays have shown some ability to predict knownnephrotoxicity of some small molecule or polypeptide drug substancessuch as amphotericin B (antifungal agent), colistin (polypeptideantibiotic), ciclosporin (immonosupressant agent), cisplatin(chemotherapeutic agent), doxorubicin (chemotherapeutic agent),gentamicin (anti-bacterial agent).

This has however not been the case for a different class of drugs namelynucleic acid based drugs such as iRNAs, antisense oligonucleotides andaptamers. Nephrotoxicity of individual nucleic acid compounds has beenpublished previously and appears to be unpredictable (see for exampleHenry et al 1997 Toxicology Vol 120 pp 145; Monteith et al 1999Toxicologic pathology Vol 27, pp. 307; Herrington et al 2011 Americanjournal of kidney diseases: the official journal of the National KidneyFoundation Vol 57, pp300; Voit et al. 2014 The Lancet. Neurology Vol 13,pp987; van Poelgeest et al 2015 British journal of clinical pharmacologyVol 80, pp1350).

To our knowledge an in vitro cell based assay using EGFR as a biomarkerfor the prediction of nephrotoxicity of a nucleic acid molecule has notbeen described. In particular the in vitro prediction of nephrotoxicityof antisense oligonucleotides has not previously been described.

OBJECTIVE OF THE INVENTION

The present invention establishes EGFR as a reliable biomarker in an invitro cell based assay for prediction of in vivo nephrotoxicity of anucleic acid molecule, such as an antisense oligonucleotide.

Reliable in vitro predictions of nephrotoxicity would increase thesuccessful clinical development of drugs, and without the use of animalsfor the initial screening drug discovery will be more cost-effective,efficient and ethical, reducing the number of animals needed fortoxicity screening of libraries of nucleic acid molecules significantly.

SUMMARY OF THE INVENTION

The invention provides in vitro toxicity assays which have been found tobe predictive for in vivo nephrotoxicity of nucleic acid molecules, inparticular oligonucleotides, such as antisense oligonucleotides.

In one aspect of the invention, the present inventors have identifiedepidermal growth factor receptor (EGFR) as a biomarker ofnephrotoxicity. This biomarker can be combined with other biomarkerssuch as kidney injury molecule-1 (KIM-1).

The invention provides for an in vitro method (an assay) for predictingin vivo toxicity (or in vivo toxicity potential) which may be used toselect nucleic acid molecule compounds which are, or are predicted tobe, suitable for in vivo administration without adverse nephrotoxicity,such as acute kidney injury or drug-induced kidney injury.

The invention provides for a method for predicting the in vivonephrotoxicity of a nucleic acid molecule, in particular a nucleic acidbased molecule such as an antisense oligonucleotide in a mammal, saidmethod comprising the steps of:

-   -   a) culturing cells expressing epidermal growth factor receptor        (EGFR) in a suitable cell culture media;    -   b) administering the nucleic acid molecule to said cell culture;    -   c) incubating the cells for a period of time; and    -   d) subsequently measuring the EGFR mRNA level in the cells;    -   wherein a decrease in EGFR mRNA is indicative of a nucleic acid        molecule which is, or is predicted to be, associated with        nephrotoxicity

The invention provides for a method for selecting one or more nucleicacid molecules suitable for in vivo administration, from a library ofnucleic acid molecules, said method comprising the steps of

-   -   a. obtaining a library of nucleic acid molecules;    -   b. administering each member of the library of nucleic acid        molecules to cell culture expressing epidermal growth factor        receptor (EGFR);    -   c. culturing the cells in vitro for a period of time;    -   d. measuring the amount of intracellular EGFR mRNA for each        nucleic acid molecule; and    -   e. selecting one or more nucleic acid molecules wherein the        reduction in EGFR relative to a control sample is above 80%.

Optionally, the method may further comprise the step of administeringthe selected nucleic acid molecule in vivo to a mammal.

Suitably, in the methods of the invention the level or amount of the atleast one biomarker may be compared to the level obtained whenadministering a control sample such as vehicle sample or a non-toxicreference nucleic acid molecule (confirmed as non-toxic in vivo) todetermine the level of increase or decrease of the at least onebiomarker due to the administration of the nucleic acid molecule (i.e.an alteration of the at least one biomarker of nephrotoxicity).

Furthermore, the at least one biomarker may be compared to the levelobtained when administering a toxic reference nucleic acid molecule(confirmed as toxic in vivo) to determine the assay window of the atleast one biomarker.

In some embodiments the cells expressing EGFR is selected from cellcultures originating from epithelial cells, mesenchymal cells andneuroectodermal cells. In particular cell cultures originating fromkidney epithelial cells are useful in the methods of the inventions,such as human PTEC or human PTEC-TERT-1 cell cultures.

In some embodiments, the predicted nephrotoxicity is associated with adecrease of EGFR biomarker in the cells. In further embodiments anincrease in KIM-1 levels are indicative of a nucleic acid molecule whichis, or is predicted to be, associated with nephrotoxicity. Furtherbiomarkers for predicting nephrotoxicity may be associated with adecrease in intracellular ATP levels.

The invention provides for the use of an in vitro assay to determine the(e.g. likely) nephrotoxicity of a nucleic acid molecule, in particular anucleic acid based molecule, such as an oligonucleotide, such as a LNAoligonucleotide. The invention provides for a nucleic acid moleculeobtained by the method for predicting nephrotoxicity of the presentinvention or by the method for screening a library of nucleic acidmolecules.

A pharmaceutical composition comprising the nucleic acid moleculeobtained by the method for predicting nephrotoxicity of the presentinvention or by the method for screening a library of nucleic acidmolecules and a pharmaceutically acceptable diluent, solvent, carrier,salt and/or adjuvant.

The nucleic acid molecule obtained by the method for predictingnephrotoxicity of the present invention or by the method for screening alibrary of nucleic acid molecules or the pharmaceutical compositioncomprising such a molecule for use in a medicine.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows morphological changes of PTEC-TERT1 cells treated with 100μM oligonucleotide for 7 days.

FIG. 2 Schematically illustrates how the EGFR and KIM-1 biomarkerscorrelate with the toxicity observed in vivo. White indicates notoxicity (innocuous); light gray indicate mild toxicity; intermediategray indicate medium toxicity; and dark gray indicate high toxicity.

DEFINITIONS

Immortalized Cell Line

The term “immortalized cell line” in the context of the presentinvention is to be understood as a population of cells descended from asingle cell in a multicellular organism, where the cells have beenmodified to escape normal cellular senescence to allow continuousproliferation or division of the cells. Immortalized cells can be grownfor prolonged periods in vitro. The mutations required for immortalitycan occur naturally or be intentionally induced e.g. using viralvectors, deletion of genes, induction of genes or proteins such astelomerases or fusion with immortal cells such as cancer cells.

Primary Cell Culture

The term “primary cell culture” in the context of the present inventionis to be understood as a population of cells isolated from a specifictissue in an animal. The primary cell culture is cultivated withoutprior genetic manipulations or clonal selection, it may however bepurified using e.g. FACS or selective growth conditions. The primarycell culture can either be fresh or established from cryopreservedtissue.

Target Nucleic Acid

According to the present invention, the target nucleic acid can be anucleic acid which encodes a mammalian protein and may for example be agene, a RNA, a mRNA, and pre-mRNA, a mature mRNA or a cDNA sequence. Thetarget nucleic acid can also be a microRNA, a long-non-coding RNA, asmall nucleolar RNA or a transfer RNA.

Nucleic Acid Molecule

The term “nucleic acid molecule” or “therapeutic nucleic acid molecule”as used herein is defined as it is generally understood by the skilledperson as a molecule comprising two or more covalently linkednucleosides. The nucleic acid molecule(s) referred to in the method ofthe invention are generally therapeutic oligonucleotides below 50nucleotides in length. The nucleic acid molecules may be or comprise anantisense oligonucleotide, or may be another oligomeric nucleic acidmolecule, such as a RNAi agent, an aptamer, or a ribozyme. Nucleic acidmolecules are commonly made in the laboratory by solid-phase chemicalsynthesis followed by purification. When referring to a sequence of thenucleic acid molecule, reference is made to the sequence or order ofnucleobase moieties, or modifications thereof, of the covalently linkednucleotides or nucleosides. The nucleic acid molecule of the inventionis man-made, and is chemically synthesized, and is typically purified orisolated. The nucleic acid molecule of the invention may comprise one ormore modified nucleosides or nucleotides.

In some embodiments, the nucleic acid molecule of the inventioncomprises or consists of 8 to 40 nucleotides in length, such as from 9to 35, such as from 10 to 30, such as from 11 to 22, such as from 12 to20, such as from 13 to 18 or 14 to 16 contiguous nucleotides in length.

In some embodiments, the nucleic acid molecule or contiguous nucleotidesequence thereof comprises or consists of 22 or less nucleotides, suchas 20 or less nucleotides, such as 18 or less nucleotides, such as 14,15, 16 or 17 nucleotides. It is to be understood that any range givenherein includes the range endpoints. Accordingly, if a nucleic acidmolecule is said to include from 10 to 30 nucleotides, both 10 and 30nucleotides are included.

In some embodiments, the contiguous nucleotide sequence comprises orconsists of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29 or 30 contiguous nucleotides in length

The nucleic acid molecule(s) are typically for modulating the expressionof one or more target nucleic acids in a mammal. In some embodiments thenucleic acid molecules, such as for siRNAs and antisenseoligonucleotides, are typically for inhibiting the expression of an RNAin a mammal, such as a mRNA or microRNA, for example. The nucleic acidmolecules may therefore be effective at modulating the expression of oneor more target nucleic acids in a mammal.

In one embodiment of the invention the nucleic acid molecule is selectedfrom a RNAi agents, an antisense oligonucleotide or an aptamer.

In some embodiment the nucleic acid molecule is an antisenseoligonucleotide.

In some embodiments the nucleic acid molecule is a phosphorothioatenucleic acid molecule.

In some embodiments the nucleic acid molecule comprises phosphorothioateinternucleoside linkages.

In some embodiments the nucleic acid molecule(s) may be conjugated tonon-nucleosidic moieties (conjugate moieties).

In some embodiments the nucleic acid molecules used or identified in themethod of the invention comprise at least one stereodefinedphosphorothioate internucleoside linkage.

A library of nucleic acid molecules is to be understood as a collectionof variant nucleic acid molecules. The purpose of the library of nucleicacid molecules can vary. In some embodiments, the library of nucleicacid molecules is composed of oligonucleotides with different nucleobasesequences, for example it may be a library of nucleic acid moleculeswhich are designed across a target nucleic acid (e.g. a RNA sequence),for example a library of antisense oligonucleotides or RNAi agentsgenerated by a mRNA gene-walk with the purpose of identifying regions onthe target nucleic acid where nucleic acid molecules efficientlymodulate the target nucleic acid. In some embodiments, the library ofnucleic acid molecules is composed of oligonucleotides with overlappingnucleobase sequence targeting a specific region on the target nucleicacid with the purpose of identifying the most potent sequence within thelibrary of nucleic acid molecules. In some embodiments, the library ofnucleic acid molecules is a library of nucleic acid molecule designvariants (child nucleic acid molecules) of a parent or ancestral nucleicacid molecule, wherein the nucleic acid molecule design variantsretaining the core nucleobase sequence of the parent nucleic acidmolecule. In some embodiments the library of nucleic acid moleculevariants (child nucleic acid molecules) differs from the parent nucleicacid molecule in one or more design parameters. The purpose of such alibrary is to improve the parent nucleic acid molecule, for example inthe context of the present application the parent nucleic acid moleculehas been found to elicit nephrotoxicity in vivo.

Antisense Oligonucleotides

The term “Antisense oligonucleotide” as used herein is defined asnucleic acid molecules capable of modulating expression of a target geneby hybridizing to a target nucleic acid, in particular to a contiguoussequence on a target nucleic acid. Antisense oligonucleotides generallycontain one or more stretches of DNA or DNA-like nucleosides. Theantisense oligonucleotides are not essentially double stranded and aretherefore not siRNAs. Preferably, the antisense oligonucleotides of thepresent invention are single stranded.

In some embodiments, the antisense oligonucleotide(s) are capable ofrecruiting RNaseH, and may, for example be a gapmer oligonucleotide asdefined herein, comprising one or more 2′ sugar modified nucleosides inthe flanks, such as 2′-O-alkyl-RNA, 2′-O-methyl-RNA, 2′-alkoxy-RNA,2′-O-methoxyethyl-RNA, 2′-amino-DNA, 2′-fluoro-DNA, arabino nucleic acid(ANA), 2′-fluoro-ANA and LNA nucleosides or mixtures of these (mixedwing gapmer), or may be a gap-breaker oligonucleotide.

In some embodiments, the antisense oligonucleotides are mixmers. Mixmeroligonucleotides typically comprise alternating regions of high affinity2′ sugar modified nucleosides, such as 2′-O-alkyl-RNA, 2′-O-methyl-RNA,2′-alkoxy-RNA, 2′-O-methoxyethyl-RNA, 2′-amino-DNA, 2′-fluoro-DNA,arabino nucleic acid (ANA), 2′-fluoro-ANA and LNA nucleosides, withshort regions of 1-4 or 1-3 DNA nucleosides. Typically a mixmer willcomprise alternating regions with short stretches of DNA, for example[LNA]₁₋₅[DNA]₁₋₃[LNA]₁₋₄[DNA]₁₋₃[LNA]₁₋₄[DNA]₁₋₃.

Various mixmer designs are highly effective, for example when targetingmicroRNA (antimiRs), microRNA binding sites on mRNAs (Blockmirs) or assplice switching oligomers (ASOs). See for example WO2007/112754(LNA-AntimiRsTM), WO2008/131807 (LNA splice switching oligos).

In some embodiments, the oligonucleotide may be a TINY LNAoligonucleotide of 7-10 nucleotides in length. Such TINY LNAs aredisclosed in WO2009/043353, herein incorporated by reference. They aretypically use to inhibit microRNAs and microRNA families, and may befull LNA modified (i.e. each nucleoside is a LNA nucleoside). It is alsopreferred that as with gapmer and mixmer oligonucleotides, theinternucleoside linkages comprise phosphorothioate internucleosidelinkages, and as with the oligonucleotides referred to herein may befully phosphorothiolates oligonucleotides.

Antisense oligonucleotides are typically between 7-30 nucleotides inlength, such as between 7-10 nucleotides (e.g. TINY LNAs) or 10-14nucleotides (e.g. shortmers or short gapmers) or 12-20 or 10-22 or 10-24nucleotides in length.

iRNA

As used herein, the terms “RNAi”, “RNAi agent,” “iRNA agent”, “RNAinterference agent” or “siRNA” as used interchangeably herein, refer toa oligonucleotide molecule that contains RNA nucleosides and whichmediates the targeted cleavage of a target nucleic acid, such as an RNAtranscript, via an RNA-induced silencing complex (RISC) pathway. iRNAdirects the sequence-specific degradation of mRNA through a processknown as RNA interference (RNAi). The iRNA modulates, e.g., inhibits,the expression of the target nucleic acid in a cell, e.g., a cell withina subject, such as a mammalian subject.

The RNAi agent subjected to the method of the invention can either be adouble stranded RNA (dsRNA) or a single stranded RNA molecule thatinteracts with a target nucleic acid sequence, such as a target RNAsequence, via the RISC pathway to direct the cleavage of the targetnucleic acid. Double stranded RNAi agents are generally between 20 and50 nucleotides in length, such as between 25 and 35 nucleotides inlength, and interacts with the endonuclease known as Dicer which isbelieved to processes dsRNA into 19-23 base pair short interfering RNAswith characteristic two base 3′ overhangs which are then incorporatedinto an RNA-induced silencing complex (RISC). Double stranded RNAiagents may siRNA molecules composed of a sense stand and and antisensestrand forming a duplex together. Alternatively it can be anoligonucleotide that forms a secondary hairpin structure whichessentially makes it double stranded in the region of the hairpin, suchmolecules are also termed shRNA's. Effective extended forms of Dicersubstrates have been described in U.S. Pat. Nos. 8,349,809 and8,513,207, hereby incorporated by reference. Upon binding to theappropriate target mRNA, one or more endonucleases within the RISCcleave the target to induce silencing. Single-stranded RNAi agents bindto the RISC endonuclease Argonaute 2, which then cleaves the targetmRNA. Single-stranded iRNAs are generally 15-30 nucleotides long and arechemically modified, e.g. including modified internucleoside linkagesand potentially also modified nucleosides. The design and testing ofsingle-stranded siRNAs are described in U.S. Pat. No. 8,101,348 and inLima et al., (2012) Cell 150: 883-894, hereby incorporated by reference.dsRNA's may be chemically modified in the same manner as the singlestranded RNAi agents.

Aptamer

As used herein, the term aptamer refers to an oligonucleotide or peptidethat forms a three-dimensional structure capable of modulating a targetthrough a ligand-target interaction, such as a ligand-proteininteraction or a ligand-DNA helix interaction. Oligonucleotide aptamerscan be formed of DNA, RNA or modified nucleosides or a mixture of these.Aptamers are effective via their three dimensional structure not throughtarget hybridization as for antisense oligonucleotides or RNAi agents.

Modified Internucleoside Linkages

Modified internucleoside linkages may, for example, be selected from thegroup comprising phosphorothioate, diphosphorothioate andboranophosphate. In some embodiments, the modified internucleosidelinkages are compatible with the RNaseH recruitment of theoligonucleotide to be tested in the method of the invention, for examplephosphorothioate, diphosphorothioate or boranophosphate.

In some embodiments the internucleoside linkage comprises sulphur (S),such as a phosphorothioate internucleoside linkage. In some embodimentsthe oligonucleotides used or identified in the method of the inventioncomprise at least one stereodefined phosphorothioate internucleosidelinkage.

A phosphorothioate internucleoside linkage is particularly useful due tonuclease resistance, beneficial pharmakokinetics and ease ofmanufacture. In some embodiments at least 50% of the internucleosidelinkages in the oligonucleotide, or contiguous nucleotide sequencethereof, are phosphorothioate, such as at least 60%, such as at least70%, such as at least 80 or such as at least 90% of the internucleosidelinkages in the oligonucleotide, or contiguous nucleotide sequencethereof, are phosphorothioate. In some embodiments all of theinternucleoside linkages of the oligonucleotide, or contiguousnucleotide sequence thereof, are phosphorothioate.

In some embodiments, the oligonucleotide comprises one or more neutralinternucleoside linkage, particularly an internucleoside linkageselected from phosphotriester, methylphosphonate, MMI, amide-3,formacetal or thioformacetal.

Further internucleoside linkages are disclosed in WO2009/124238(incorporated herein by reference). In an embodiment the internucleosidelinkage is selected from linkers disclosed in WO2007/031091(incorporated herein by reference). Particularly, the internucleosidelinkage may be selected from —O—P(O)₂—O—, —O—P(O,S)—O—, —O—P(S)₂—O—,—S—P(O)₂—O—, —S—P(O,S)—O—, —S—P(S)₂—O—, —O—P(O)₂—S—, —O—P(O,S)—S—,—S—P(O)₂—S—, —O—PO(RH)—O—, O—PO(OCH₃)—O—, —O—PO(NRH)—O—,—O—PO(OCH₂CH₂S—R)—O—, —O—PO(BH₃)—O—, —O—PO(NHRH)—O—, —O—P(O)₂—NRH—,—NRH—P(O)₂—O—, —NRH—CO—O—, —NRH—CO—NRH—, and/or the internucleosidelinker may be selected form the group consisting of: —O—CO—O—,—O—CO—NRH—, —NRH—CO—CH₂—, —O—CH₂—CO—NRH—, —O—CH₂—CH₂—NRH—, —CO—NRH—CH₂—,—CH₂—NRHCO—, —O—CH₂—CH₂—S—, —S—CH₂—CH₂—O—, —S—CH₂—CH₂—S—, —CH₂—SO₂—CH₂—,—CH₂—CO—NRH—, —O—CH₂—CH₂—NRH—CO—, —CH₂—NCH₃—O—CH₂—, where RH is selectedfrom hydrogen and C₁₋₄-alkyl.

Nuclease resistant linkages, such as phosphothioate linkages, areparticularly useful in oligonucleotide regions capable of recruitingnuclease when forming a duplex with the target nucleic acid, such asregion G for gapmers, or the non-modified nucleoside region of headmersand tailmers. Phosphorothioate linkages may, however, also be useful innon-nuclease recruiting regions and/or affinity enhancing regions suchas regions F and F′ for gapmers, or the modified nucleoside region ofheadmers and tailmers.

Each of the design regions may however comprise internucleoside linkagesother than phosphorothioate, such as phosphodiester linkages, inparticularly in regions where modified nucleosides, such as LNA, protectthe linkage against nuclease degradation. Inclusion of phosphodiesterlinkages, such as one or two linkages, particularly between or adjacentto modified nucleoside units (typically in the non-nuclease recruitingregions) can modify the bioavailability and/or bio-distribution of anoligonucleotide—see WO2008/113832, incorporated herein by reference.

In an embodiment all the internucleoside linkages in the oligonucleotideare phosphorothioate and/or boranophosphate linkages. In someembodiment, all the internucleoside linkages in the oligonucleotide arephosphorothioate linkages.

Stereodefined Internucleotide Linkages

In the context of the present invention the term “stereodfined” refersto nucleic acid molecules, such as oligonucleotides including antisense,RNI and aptamer molecules, where at least one phosphorothioateinternucleoside linkage present in the oligonucleotide has definedstereochemistry, i.e. either Rp or Sp. In some embodiments all of thephosphorothioate internucleoside linkages in a stereodefinedoligonucleotide may be stereodefined, i.e. each phosphorothioateinternucleoside linkage is independently selected from the groupconsisting of Rp and Sp phosphorothioate internucleoside linkages.

Typically, oligonucleotide phosphorothioates are synthesised as a randommixture of Rp and Sp phosphorothioate linkages (also referred to as aracemic mixture). In the present invention, gapmer phosphorothioateoligonucleotides are provided where at least one of the phosphorothioatelinkages of the gap region oligonucleotide is stereodefined, i.e. iseither Rp or Sp in at least 75%, such as at least 80%, or at least 85%,or at least 90% or at least 95%, or at least 97%, such as at least 98%,such as at least 99%, or (essentially) all of the oligonucleotidemolecules present in the oligonucleotide sample. Such oligonucleotidesmay be referred as being stereodefined, stereoselective orstereospecified: They comprise at least one phosphorothioate linkagewhich is stereospecific. The terms stereodefined andstereospecified/stereoselective may be used interchangeably herein. Theterms stereodefined, stereoselective and stereospecified may be used todescribe a phosphorothioate internucleoside linkage (Rp or Sp), or maybe used to described a oligonucleotide which comprises such aphosphorothioate internucleoside linkage. It is recognized that astereodefined oligonucleotide may comprise a small amount of thealternative stereoisomer at any one position, for example Wan et alreports a 98% stereoselectivity for the gapmers reported in NAR,November 2014.

Modulation of Expression

The term “modulation of expression” as used herein is to be understoodas an overall term for an oligonucleotide's ability to alter the amountof a nucleic acid target when compared to the amount of the nucleic acidtarget before administration of the oligonucleotide. Alternativelymodulation of expression may be determined by reference to a controlexperiment. It is generally understood that the control is an individualor target cell treated with a saline composition or an individual ortarget cell treated with a non-targeting oligonucleotide (mock). It mayhowever also be an individual treated with the standard of care.

One type of modulation is an oligonucleotide's ability to inhibit,down-regulate, reduce, suppress, remove, stop, block, prevent, lessen,lower, avoid or terminate expression of the nucleic acid target e.g. bydegradation of mRNA or blockage of transcription. Another type ofmodulation is an oligonucleotide's ability to restore, increase orenhance expression of nucleic acid target e.g. by repair of splice sitesor prevention of splicing or removal or blockage of inhibitorymechanisms such as microRNA repression.

In some embodiments of the invention, when the target of theoligonucleotide of the invention is present in the EGFR expressingcells, the method of the invention may further comprise the step ofdetermining the level of target modulation (e.g. inhibit for siRNAs orantisense oligonucleotides) in the population of EGFR expressing cellsafter treatment with the oligonucleotides (e.g. this may occur inparallel or as part of the measurement of the at least one biomarkerstep). In this regard the method of the invention may be used todetermine the comparative potency or effectiveness of theoligonucleotide and the comparative toxicity, allowing for the selectionof potent non-toxic compounds for use in vivo. It will be understoodthat the determination of compound potency/effectiveness may beperformed in a separate in vitro experiment, either in the EGFRexpressing cells, particularly cells which are expressing the target.

Modified Oligonucleotides

Non modified DNA and RNA molecules are rapidly degraded in vivo, and assuch are of little use therapeutically. Typically, theoligonucleotide(s) used in the method of the invention are thereforemodified. One widely used modification is the use of phosphorothioateinternucleoside linkages, which is known to stabilise oligonucleotidesfrom nucleolytic degradation, as well as providing desirablepharmacological properties. In some embodiments the oligonucleotide(s)comprise phosphorothioate internucleoside linkages.

Another desirable modification are those which confer higher affinity ofthe oligonucleotide to the target nucleic acid, so called high affinitymodified nucleotides, which include bicyclic “LNA” nucleosides as wellas numerous 2′ substituted nucleosides.

High Affinity Modified Nucleosides

In some embodiments, the oligonucleotide comprises one or more highaffinity modified nucleoside. A high affinity modified nucleoside is amodified nucleoside which, when incorporated into the oligonucleotideenhances the affinity of the oligonucleotide for its complementarytarget, for example as measured by the melting temperature (T^(m)). Ahigh affinity modified nucleoside of the present invention preferablyresult in an increase in melting temperature between +0.5 to +12° C.,more preferably between +1.5 to +10° C. and most preferably between +3to +8° C. per modified nucleoside. Numerous high affinity modifiednucleosides are known in the art and include for example 2′ sugarmodified nucleosides, such as many 2′ substituted nucleosides as well aslocked nucleic acids (LNA) (see e.g. Freier & Altmann; Nucl. Acid Res.,1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development,2000, 3(2), 293-213).

Sugar Modifications

The oligonucleotide(s) may comprise one or more nucleosides which have amodified sugar moiety, i.e. a modification of the sugar moiety whencompared to the ribose sugar moiety found in naturally occurring DNA andRNA nucleosides.

Numerous nucleosides with modification of the ribose sugar moiety havebeen made, primarily with the aim of improving certain properties ofoligonucleotides, such as affinity and/or nuclease resistance. Suchmodifications include those where the ribose ring structure is modified,e.g. by replacement with a hexose ring (HNA).

Sugar modifications also include modifications made via altering thesubstituent groups on the ribose ring to groups other than hydrogen, orthe 2′-OH group naturally found in DNA and RNA nucleosides. Substituentsmay, for example be introduced at the 2′, 3′, 4′ or 5′ positions of theribose ring. Nucleosides with modified sugar moieties also include 2′modified nucleosides, such as 2′ substituted nucleosides. Numerous 2′substituted nucleosides have been found to have beneficial propertieswhen incorporated into oligonucleotides, such as enhanced nucleosideresistance and enhanced affinity. In one embodiment the nucleic acidmolecule(s) or library of such molecules comprises one or more 2′ sugarmodified nucleoside.

In addition to the 2′ substitution there are other modificationsincluding a modification at position 2 of the ribose ring, such asintroduction of a bicyclic ring, which typically have a biradicle bridgebetween the C2 and C4 carbons on the ribose ring (also known as lockednucleic acid or LNA), or an unlinked ribose ring which typically lacks abond between the C2 and C3 carbons (e.g. UNA). Other sugar modifiednucleosides include, for example, bicyclohexose nucleic acids(WO2011/017521) or tricyclic nucleic acids (WO2013/154798). Modifiednucleosides also include nucleosides where the sugar moiety is replacedwith a non-sugar moiety, for example in the case of peptide nucleicacids (PNA), or morpholino nucleic acids.

2′Sugarmodified Nucleosides.

A 2′ sugar modified nucleoside is a nucleoside which has a substituentother than H or —OH at the 2′ position (2′ substituted nucleoside) orcomprises a 2′ linked biradicle, and includes 2′ substituted nucleosidesand LNA (2′-4′ biradicle bridged) nucleosides. For example, the 2′modified sugar may provide enhanced binding affinity and/or increasednuclease resistance to the oligonucleotide. Examples of 2′ substitutedmodified nucleosides are 2′-O-alkyl-RNA, 2′-O-methyl-RNA, 2′-alkoxy-RNA,2′-O-methoxyethyl-RNA (MOE), 2′-amino-DNA, 2′-Fluoro-RNA, and 2′-F-ANAnucleoside. For further examples, please see e.g. Freier & Altmann;Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in DrugDevelopment, 2000, 3(2), 293-213, and Deleavey and Damha, Chemistry andBiology 2012, 19, 937. Below are illustrations of some 2′ substitutedmodified nucleosides.

Locked Nucleic Acid Nucleosides (LNA)

In some embodiments oligonucleotides are LNA oligonucleotides, i.e. theycomprise at least one LNA nucleoside.

LNA monomers (also referred to as bicyclic nucleic acids, BNA) arenucleosides where there is a biradical between the 2′ and 4′ position ofthe ribose ring. The 2′-4′ biradical is also referred to as a bridge.LNA monomers, when incorporated into a oligonucleotides are known toenhance the binding affinity of the oligonucleotide to a complementaryDNA or RNA sequence, typically measured or calculated as an increase inthe temperature required to melt the oligonucleotide/target duplex(T_(m)).

The LNA oligomer may be a single stranded antisense oligonucleotide.

The LNA used in the oligonucleotide compounds of the invention may havethe structure of the general formula I

-   -   wherein for all chiral centers, asymmetric groups may be found        in either R or S orientation;    -   wherein X is selected from —O—, —S—, —N(R^(N*))—, —C(R⁶R^(6*))—,        such as, in some embodiments —O—;    -   B is selected from hydrogen, optionally substituted C₁₋₄-alkoxy,        optionally substituted C₁₋₄-alkyl, optionally substituted        C₁₋₄-acyloxy, nucleobases including naturally occurring and        nucleobase analogues, DNA intercalators, photochemically active        groups, thermochemically active groups, chelating groups,        reporter groups, and ligands; preferably, B is a nucleobase or        nucleobase analogue;    -   P designates an internucleotide linkage to an adjacent monomer,        or a 5-terminal group, such internucleotide linkage or        5-terminal group optionally including the substituent R⁵ or        equally applicable the substituent R^(5*);    -   P* designates an internucleotide linkage to an adjacent monomer,        or a 3′-terminal group;    -   R^(4*) and R^(2*) together designate a bivalent linker group        consisting of 1-4 groups/atoms selected from —C(R^(a)R^(b))—,        —C(R^(a))═C(R^(b))—, —C(R^(a))═N—, —O—, —Si(R^(a))₂—, —S—,        —SO₂—, —N(R^(a))—, and >C═Z, wherein Z is selected from —O—,        —S—, and —N(R^(a))—, and R^(a) and R^(b) each is independently        selected from hydrogen, optionally substituted C₁₋₁₂-alkyl,        optionally substituted C₂₋₁₂-alkenyl, optionally substituted        C₂₋₁₂-alkynyl, hydroxy, optionally substituted C₁₋₁₂-alkoxy,        C₂₋₁₂-alkoxyalkyl, C₂₋₁₂-alkenyloxy, carboxy,        C₁₋₁₂-alkoxycarbonyl, C₁₋₁₂-alkylcarbonyl, formyl, aryl,        aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl,        heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl,        amino, mono- and di(C₁₋₆-alkyl)amino, carbamoyl, mono- and        di(C₁₋₆-alkyl)-amino-carbonyl, amino-C₁₋₆-alkyl-aminocarbonyl,        mono- and di(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl,        C₁₋₆-alkyl-carbonylamino, carbamido, C₁₋₆-alkanoyloxy, sulphono,        C₁₋₆-alkylsulphonyloxy, nitro, azido, sulphanyl, C₁₋₆-alkylthio,        halogen, DNA intercalators, photochemically active groups,        thermochemically active groups, chelating groups, reporter        groups, and ligands, where aryl and heteroaryl may be optionally        substituted and where two geminal substituents R^(a) and R^(b)        together may designate optionally substituted methylene (═CH₂),        wherein for all chiral centers, asymmetric groups may be found        in either R or S orientation, and;    -   each of the substituents R^(1*), R², R³, R⁵, R^(5*), R⁶ and        R^(6*), which are present is independently selected from        hydrogen, optionally substituted C₁₋₁₂-alkyl, optionally        substituted C₂₋₁₂-alkenyl, optionally substituted C₂₋₁₂-alkynyl,        hydroxy, C₁₋₁₂-alkoxy, C₂₋₁₂-alkoxyalkyl, C₂₋₁₂-alkenyloxy,        carboxy, C₁₋₁₂-alkoxycarbonyl, C₁₋₁₂-alkylcarbonyl, formyl,        aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl,        heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl,        amino, mono- and di(C₁₋₆-alkyl)amino, carbamoyl, mono- and        di(C₁₋₆-alkyl)-amino-carbonyl, amino-C₁₋₆-alkyl-aminocarbonyl,        mono- and di(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl,        C₁₋₆-alkyl-carbonylamino, carbamido, C₁₋₆-alkanoyloxy, sulphono,        C₁₋₆-alkylsulphonyloxy, nitro, azido, sulphanyl, C₁₋₆-alkylthio,        halogen, DNA intercalators, photochemically active groups,        thermochemically active groups, chelating groups, reporter        groups, and ligands, where aryl and heteroaryl may be optionally        substituted, and where two geminal substituents together may        designate oxo, thioxo, imino, or optionally substituted        methylene; wherein R^(N) is selected from hydrogen and        C₁₋₄-alkyl, and where two adjacent (non-geminal) substituents        may designate an additional bond resulting in a double bond; and        R^(N*), when present and not involved in a biradical, is        selected from hydrogen and C₁₋₄-alkyl; and basic salts and acid        addition salts thereof. For all chiral centers, asymmetric        groups may be found in either R or S orientation.

In some embodiments, R^(4*) and R^(2*) together designate a biradicalconsisting of a groups selected from the group consisting ofC(R^(a)R^(b))—C(R^(a)R^(b))—, C(R^(a)R^(b))—O—, C(R^(a)R^(b))—NR^(a)—,C(R^(a)R^(b))—S—, and C(R^(a)R^(b))—C(R^(a)R^(b))—O—, wherein each R^(a)and R^(b) may optionally be independently selected. In some embodiments,R^(a) and R^(b) may be, optionally independently selected from the groupconsisting of hydrogen and _(C1-6)alkyl, such as methyl, such ashydrogen.

In some embodiments, R^(4*) and R^(2*) together designate the biradical—O—CH(CH₂OCH₃)-(2′O-methoxyethyl bicyclic nucleic acid—Seth at al.,2010, J. Org. Chem)—in either the R- or S-configuration.

In some embodiments, R^(4*) and R^(2*) together designate the biradical—O—CH(CH₂CH₃)-(2′O-ethyl bicyclic nucleic acid—Seth at al., 2010, J.Org. Chem).—in either the R- or S-configuration.

In some embodiments, R^(4*) and R^(2*) together designate the biradical—O—CH(CH₃)—.—in either the R- or S-configuration. In some embodiments,R^(4*) and R^(2*) together designate the biradical —O—CH₂—O—CH₂——(Sethat al., 2010, J. Org. Chem).

In some embodiments, R^(4*) and R^(2*) together designate the biradical—O—NR—CH₃——(Seth at al., 2010, J. Org. Chem).

In some embodiments, the LNA units have a structure selected from thefollowing group:

In some embodiments, R^(1*), R², R³, R⁵, R^(5*) are independentlyselected from the group consisting of hydrogen, halogen, C₁₋₆ alkyl,substituted C₁₋₆ alkyl, C₂₋₆ alkenyl, substituted C₂₋₆ alkenyl, C₂₋₆alkynyl or substituted C₂₋₆ alkynyl, C₁₋₆ alkoxyl, substituted C₁₋₆alkoxyl, acyl, substituted acyl, C₁₋₆ aminoalkyl or substituted C₁₋₆aminoalkyl. For all chiral centers, asymmetric groups may be found ineither R or S orientation.

In some embodiments, R^(1*), R², R³, R⁵, R^(5*) are hydrogen.

In some embodiments, R^(1*), R², R³ are independently selected from thegroup consisting of hydrogen, halogen, C₁₋₆ alkyl, substituted C₁₋₆alkyl, C₂₋₆ alkenyl, substituted C₂₋₆ alkenyl, C₂₋₆ alkynyl orsubstituted C₂₋₆ alkynyl, C₁₋₆ alkoxyl, substituted C₁₋₆ alkoxyl, acyl,substituted acyl, C₁₋₆ aminoalkyl or substituted C₁₋₆ aminoalkyl. Forall chiral centers, asymmetric groups may be found in either R or Sorientation.

In some embodiments, R^(1*), R², R³ are hydrogen.

In some embodiments, R⁵ and R^(5*) are each independently selected fromthe group consisting of H, —CH₃, —CH₂—CH₃, —CH₂—O—CH₃, and —CH═CH₂.Suitably in some embodiments, either R⁵ or R^(5*) are hydrogen, where asthe other group (R⁵ or R^(5*) respectively) is selected from the groupconsisting of C₁₋₅ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, substituted C₁₋₆alkyl, substituted C₂₋₆ alkenyl, substituted C₂₋₆ alkynyl or substitutedacyl (—C(═O)—); wherein each substituted group is mono or polysubstituted with substituent groups independently selected from halogen,C₁₋₆ alkyl, substituted C₁₋₆ alkyl, C₂₋₆ alkenyl, substituted C₂₋₆alkenyl, C₂₋₆ alkynyl, substituted C₂₋₆ alkynyl, OJ₁, SJ₁, NJ₁J₂, N₃,COOJ₁, CN, O—C(═O)NJ₁J₂, N(H)C(═NH)NJ, J₂ or N(H)C(═X)N(H)J₂ wherein Xis O or S; and each J₁ and J₂ is, independently, H, C₁₋₆ alkyl,substituted C₁₋₆ alkyl, C₂₋₆ alkenyl, substituted C₂₋₆ alkenyl, C₂₋₆alkynyl, substituted C₂₋₆ alkynyl, C₁₋₆ aminoalkyl, substituted C₁₋₆aminoalkyl or a protecting group. In some embodiments either R⁵ orR^(5*) is substituted C₁₋₆ alkyl. In some embodiments either R⁵ orR^(5*) is substituted methylene wherein preferred substituent groupsinclude one or more groups independently selected from F, NJ₁J₂, N₃, CN,OJ₁, SJ₁, O—C(═O)NJ₁J₂, N(H)C(═NH)NJ, J₂ or N(H)C(O)N(H)J₂. In someembodiments each J₁ and J₂ is, independently H or C₁₋₆ alkyl. In someembodiments either R⁵ or R^(5*) is methyl, ethyl or methoxymethyl. Insome embodiments either R⁵ or R^(5*) is methyl. In a further embodimenteither R⁵ or R^(5*) is ethylenyl. In some embodiments either R⁵ orR^(5*) is substituted acyl. In some embodiments either R⁵ or R^(5*) isC(═O)NJ₁J₂. For all chiral centers, asymmetric groups may be found ineither R or S orientation. Such 5′ modified bicyclic nucleotides aredisclosed in WO 2007/134181, which is hereby incorporated by referencein its entirety.

In some embodiments B is a nucleobase, including nucleobase analoguesand naturally occurring nucleobases, such as a purine or pyrimidine, ora substituted purine or substituted pyrimidine, such as a nucleobasereferred to herein, such as a nucleobase selected from the groupconsisting of adenine, cytosine, thymine, adenine, uracil, and/or amodified or substituted nucleobase, such as 5-thiazolo-uracil,2-thio-uracil, 5-propynyl-uracil, 2′thio-thymine, 5-methyl cytosine,5-thiozolo-cytosine, 5-propynyl-cytosine, and 2,6-diaminopurine.

In some embodiments, R^(4*) and R^(2*) together designate a biradicalselected from —C(R^(a)R^(b))—O—, —C(R^(a)R^(b))—C(R^(c)R^(d))—O—,—C(R^(a)R^(b))—C(R^(c)R^(d))—C(R^(e)R^(f))—O—,—C(R^(a)R^(b))—O—C(R^(c)R^(d))—, —C(R^(a)R^(b))—O—C(R^(c)R^(d))—O—,—C(R^(a)R^(b))—C(R^(c)R^(d))—,—C(R^(a)R^(b))—C(R^(c)R^(d))—C(R^(e)R^(f))—,—C(R^(a))═C(R^(b))—C(R^(c)R^(d))—, —C(R^(a)R^(b))—N(R^(c))—,—C(R^(a)R^(b))—C(R^(c)R^(d))—N(R^(e))—, —C(R^(a)R^(b))—N(R^(c))—O—, and—C(R^(a)R^(b))—S—, —C(R^(a)R^(b))—C(R^(c)R^(d))—S—, wherein R^(a),R^(b), R^(c), R^(d), R^(e), and R^(f) each is independently selectedfrom hydrogen, optionally substituted C₁₋₁₂-alkyl, optionallysubstituted C₂₋₁₂-alkenyl, optionally substituted C₂₋₁₂-alkynyl,hydroxy, C₁₋₁₂-alkoxy, C₂₋₁₂-alkoxyalkyl, C₂₋₁₂-alkenyloxy, carboxy,C₁₋₁₂-alkoxycarbonyl, C₁₋₁₂-alkylcarbonyl, formyl, aryl,aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl,heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di(C₁₋₆-alkyl)amino, carbamoyl, mono- anddi(C₁₋₆-alkyl)-amino-carbonyl, amino-C₁₋₆-alkyl-aminocarbonyl, mono- anddi(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl, C₁₋₆-alkyl-carbonylamino,carbamido, C₁₋₆-alkanoyloxy, sulphono, C₁₋₆-alkylsulphonyloxy, nitro,azido, sulphanyl, C₁₋₆-alkylthio, halogen, DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands, where aryl and heteroaryl may beoptionally substituted and where two geminal substituents R^(a) andR^(b) together may designate optionally substituted methylene (═CH₂).For all chiral centers, asymmetric groups may be found in either R or Sorientation.

In a further embodiment R^(4*) and R^(2*) together designate a biradical(bivalent group) selected from —CH₂—O—, —CH₂—S—, —CH₂—NH—, —CH₂—N(CH₃)—,—CH₂—CH₂—O—, —CH₂—CH(CH₃)—, —CH₂—CH₂—S—, —CH₂—CH₂—NH—, —CH₂—CH₂—CH₂—,—CH₂—CH₂—CH₂—O—, —CH₂—CH₂—CH(CH₃)—, —CH═CH—CH₂—, —CH₂—O—CH₂—O—,—CH₂—NH—O—, —CH₂—N(CH₃)—O—, —CH₂—O—CH₂—, —CH(CH₃)—O—, and—CH(CH₂—O—CH₃)—O—, and/or, —CH₂—CH₂—, and —CH═CH— For all chiralcenters, asymmetric groups may be found in either R or S orientation.

In some embodiments, R^(4*) and R^(2*) together designate the biradicalC(R^(a)R^(b))—N(R^(c))—O—, wherein R^(a) and R^(b) are independentlyselected from the group consisting of hydrogen, halogen, C₁₋₆ alkyl,substituted C₁₋₆ alkyl, C₂₋₆ alkenyl, substituted C₂₋₆ alkenyl, C₂₋₆alkynyl or substituted C₂₋₆ alkynyl, C₁₋₆ alkoxyl, substituted C₁₋₆alkoxyl, acyl, substituted acyl, C₁₋₆ aminoalkyl or substituted C₁₋₆aminoalkyl, such as hydrogen, and; wherein R^(c) is selected from thegroup consisting of hydrogen, halogen, C₁₋₆ alkyl, substituted C₁₋₆alkyl, C₂₋₆ alkenyl, substituted C₂₋₆ alkenyl, C₂₋₆ alkynyl orsubstituted C₂₋₆ alkynyl, C₁₋₆ alkoxyl, substituted C₁₋₆ alkoxyl, acyl,substituted acyl, C₁₋₆ aminoalkyl or substituted C₁₋₆ aminoalkyl, suchas hydrogen.

In some embodiments, R^(4*) and R^(2*) together designate the biradicalC(R^(a)R^(b))—O—C(R^(c)R^(d))—O—, wherein R^(a), R^(b), R^(c), and R^(d)are independently selected from the group consisting of hydrogen,halogen, C₁₋₆ alkyl, substituted C₁₋₆ alkyl, C₂₋₆ alkenyl, substitutedC₂₋₆ alkenyl, C₂₋₆ alkynyl or substituted C₂₋₆ alkynyl, C₁₋₆ alkoxyl,substituted C₁₋₆ alkoxyl, acyl, substituted acyl, C₁₋₆ aminoalkyl orsubstituted C₁₋₆ aminoalkyl, such as hydrogen.

In some embodiments, R^(4*) and R^(2*) form the biradical —CH(Z)—O—,wherein Z is selected from the group consisting of C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, substituted C₁₋₆ alkyl, substituted C₂₋₆ alkenyl,substituted C₂₋₆ alkynyl, acyl, substituted acyl, substituted amide,thiol or substituted thio; and wherein each of the substituted groups,is, independently, mono or poly substituted with optionally protectedsubstituent groups independently selected from halogen, oxo, hydroxyl,OJ₁, NJ₁J₂, SJ₁, N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ³C(═X)NJ₁J₂ and CN,wherein each J₁, J₂ and J₃ is, independently, H or C₁₋₆ alkyl, and X isO, S or NJ₁. In some embodiments Z is C₁₋₆ alkyl or substituted C₁₋₆alkyl. In some embodiments Z is methyl. In some embodiments Z issubstituted C₁₋₆ alkyl. In some embodiments said substituent group isC₁₋₆ alkoxy. In some embodiments Z is CH₃OCH₂—. For all chiral centers,asymmetric groups may be found in either R or S orientation. Suchbicyclic nucleotides are disclosed in U.S. Pat. No. 7,399,845 which ishereby incorporated by reference in its entirety. In some embodiments,R^(1*), R², R³, R⁵, R^(5*) are hydrogen. In some some embodiments,R^(1*), R², R^(3*) are hydrogen, and one or both of R⁵, R^(5*) may beother than hydrogen as referred to above and in WO 2007/134181.

In some embodiments, R^(4*) and R^(2*) together designate a biradicalwhich comprise a substituted amino group in the bridge such as consistor comprise of the biradical —CH₂—N(R^(c))—, wherein R^(c) is C₁₋₁₂alkyloxy. In some embodiments R^(4*) and R^(2*) together designate abiradical —Cq₃q₄-NOR—, wherein q₃ and q₄ are independently selected fromthe group consisting of hydrogen, halogen, C₁₋₆ alkyl, substituted C₁₋₆alkyl, C₂₋₆ alkenyl, substituted C₂₋₆ alkenyl, C₂₋₆ alkynyl orsubstituted C₂₋₆ alkynyl, C₁₋₆ alkoxyl, substituted C₁₋₆ alkoxyl, acyl,substituted acyl, C₁₋₆ aminoalkyl or substituted C₁₋₆ aminoalkyl;wherein each substituted group is, independently, mono or polysubstituted with substituent groups independently selected from halogen,OJ₁, SJ₁, NJ₁J₂, COOJ₁, CN, O—C(═O)NJ₁J₂, N(H)C(═NH)N J₁J₂ orN(H)C(═X═N(H)J₂ wherein X is O or S; and each of J₁ and J₂ is,independently, H, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆aminoalkyl or a protecting group. For all chiral centers, asymmetricgroups may be found in either R or S orientation. Such bicyclicnucleotides are disclosed in WO2008/150729 which is hereby incorporatedby reference in its entirity. In some embodiments, R^(1*), R², R³, R⁵,R^(5*) are independently selected from the group consisting of hydrogen,halogen, C₁₋₆ alkyl, substituted C₁₋₆ alkyl, C₂₋₆ alkenyl, substitutedC₂₋₆ alkenyl, C₂₋₆ alkynyl or substituted C₂₋₆ alkynyl, C₁₋₆ alkoxyl,substituted C₁₋₆ alkoxyl, acyl, substituted acyl, C₁₋₆ aminoalkyl orsubstituted C₁₋₆ aminoalkyl. In some embodiments, R^(1*), R², R³, R⁵,R^(5*) are hydrogen. In some embodiments, R^(1*), R², R³ are hydrogenand one or both of R⁵, R^(5*) may be other than hydrogen as referred toabove and in WO 2007/134181. In some embodiments R^(4*) and R^(2*)together designate a biradical (bivalent group) C(R^(a)R^(b))—O—,wherein R^(a) and R^(b) are each independently halogen, C₁-C₁₂ alkyl,substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl,C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₁-C₁₂ alkoxy, substitutedC₁-C₁₂ alkoxy, OJ₁ SJ₁, SOJ₁, SO₂J₁, NJ₁J₂, N₃, CN, C(═O)OJ₁,C(═O)NJ₁J₂, C(═O)J₁, O—C(═O)NJ₁J₂, N(H)C(═NH)NJ₁J₂, N(H)C(═O)NJ₁J₂ orN(H)C(═S)NJ₁J₂; or R^(a) and R^(b) together are ═C(q₃)(q₄); q₃ and q₄are each, independently, H, halogen, C₁-C₁₂alkyl or substituted C₁-C₁₂alkyl; each substituted group is, independently, mono or polysubstituted with substituent groups independently selected from halogen,C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl, OJ₁, SJ₁, NJ₁J₂, N₃,CN, C(═O)OJ₁, C(═O)NJ₁J₂, C(═O)J₁, O—C(═O)NJ₁J₂, N(H)C(═O)NJ₁J₂ orN(H)C(═S)NJ₁J₂ and; each J₁ and J₂ is, independently, H, C1-C₆ alkyl,substituted C1-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl, substituted C₂-C₆ alkynyl, C1-C₆ aminoalkyl, substituted C1-C₆aminoalkyl or a protecting group. Such compounds are disclosed inWO2009006478A, hereby incorporated in its entirety by reference.

In some embodiments, R^(4*) and R^(2*) form the biradical -Q-, wherein Qis C(q₁)(q₂)C(q₃)(q₄), C(q₁)=C(q₃), C[═C(q₁)(q₂)]-C(q₃)(q₄) orC(q₁)(q₂)-C[═C(q₃)(q₄)]; q₁, q₂, q₃, q₄ are each independently. H,halogen, C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl,substituted C₁₋₁₂ alkoxy, OJ₁, SJ₁, SOJ₁, SO₂J₁, NJ₁J₂, N₃, CN,C(═O)OJ₁, C(═O)—NJ₁J₂, C(═O) J₁, —C(═O)NJ₁J₂, N(H)C(═NH)NJ₁J₂,N(H)C(═O)NJ₁J₂ or N(H)C(═S)NJ₁J₂; each J₁ and J₂ is, independently, H,C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆ aminoalkyl or a protectinggroup; and, optionally wherein when Q is C(q₁)(q₂)(q₃)(q₄) and one of q₃or q₄ is CH₃ then at least one of the other of q₃ or q₄ or one of q₁ andq₂ is other than H. In some embodiments, R^(1*), R², R³, R⁵, R^(5*) arehydrogen. For all chiral centers, asymmetric groups may be found ineither R or S orientation. Such bicyclic nucleotides are disclosed inWO2008/154401 which is hereby incorporated by reference in its entirity.In some embodiments, R^(1*), R², R³, R⁵, R^(5*) are independentlyselected from the group consisting of hydrogen, halogen, C₁₋₆ alkyl,substituted C₁₋₆ alkyl, C₂₋₆ alkenyl, substituted C₂₋₆ alkenyl, C₂₋₆alkynyl or substituted C₂₋₆ alkynyl, C₁₋₆ alkoxyl, substituted C₁₋₆alkoxyl, acyl, substituted acyl, C₁₋₆ aminoalkyl or substituted C₁₋₆aminoalkyl. In some embodiments, R^(1*), R², R³, R⁵, R^(5*) arehydrogen. In some embodiments, R^(1*), R², R³ are hydrogen and one orboth of R⁵, R^(5*) may be other than hydrogen as referred to above andin WO 2007/134181 or WO2009/067647 (alpha-L-bicyclic nucleic acidsanalogs).

Further bicyclic nucleoside analogues and their use in antisenseoligonucleotides are disclosed in WO2011 115818, WO2011/085102,WO2011/017521, WO09100320, WO10036698, WO09124295 & WO09006478. Suchnucleoside analogues may in some aspects be useful in the compounds ofpresent invention.

In some embodiments the LNA used in the oligonucleotide compounds of theinvention preferably has the structure of the general formula II:

wherein Y is selected from the group consisting of —O—, —CH₂O—, —S—,—NH—, N(R^(e)) and/or —CH₂—; Z and Z* are independently selected amongan internucleotide linkage, RH, a terminal group or a protecting group;B constitutes a natural or non-natural nucleotide base moiety(nucleobase), and RH is selected from hydrogen and C₁₋₄-alkyl; R^(a),R^(b) R^(c), R^(d) and R^(e) are, optionally independently, selectedfrom the group consisting of hydrogen, optionally substitutedC₁₋₁₂-alkyl, optionally substituted C₂₋₁₂-alkenyl, optionallysubstituted C₂₋₁₂-alkynyl, hydroxy, C₁₋₁₂-alkoxy, C₂₋₁₂-alkoxyalkyl,C₂₋₁₂-alkenyloxy, carboxy, C₁₋₁₂-alkoxycarbonyl, C₁₋₁₂-alkylcarbonyl,formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl,heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di(C₁₋₆-alkyl)amino, carbamoyl, mono- anddi(C₁₋₆-alkyl)-amino-carbonyl, amino-C₁₋₆-alkyl-aminocarbonyl, mono- anddi(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl, C₁₋₆-alkyl-carbonylamino,carbamido, C₁₋₆-alkanoyloxy, sulphono, C₁₋₆-alkylsulphonyloxy, nitro,azido, sulphanyl, C₁₋₆-alkylthio, halogen, DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands, where aryl and heteroaryl may beoptionally substituted and where two geminal substituents R^(a) andR^(b) together may designate optionally substituted methylene (═CH₂);and RH is selected from hydrogen and C₁₋₄-alkyl. In some embodimentsR^(a), R^(b) R^(c), R^(d) and R^(e) are, optionally independently,selected from the group consisting of hydrogen and C₁₋₆ alkyl, such asmethyl. For all chiral centers, asymmetric groups may be found in eitherR or S orientation, for example, two exemplary stereochemical isomersinclude the beta-D and alpha-L isoforms, which may be illustrated asfollows:

Specific exemplary LNA units are shown below:

The term “thio-LNA” comprises a locked nucleotide in which Y in thegeneral formula above is selected from S or —CH₂—S—. Thio-LNA can be inboth beta-D and alpha-L-configuration.

The term “amino-LNA” comprises a locked nucleotide in which Y in thegeneral formula above is selected from —N(H)—, N(R)—, CH₂—N(H)—, and—CH₂—N(R)— where R is selected from hydrogen and C₁₋₄-alkyl. Amino-LNAcan be in both beta-D and alpha-L-configuration.

The term “oxy-LNA” comprises a locked nucleotide in which Y in thegeneral formula above represents —O—. Oxy-LNA can be in both beta-D andalpha-L-configuration.

The term “ENA” comprises a locked nucleotide in which Y in the generalformula above is —CH₂—O— (where the oxygen atom of —CH₂—O— is attachedto the 2′-position relative to the base B). R^(e) is hydrogen or methyl.

In some exemplary embodiments LNA is selected from beta-D-oxy-LNA,alpha-L-oxy-LNA, beta-D-amino-LNA and beta-D-thio-LNA, in particularbeta-D-oxy-LNA.

Certain examples of LNA nucleosides are presented in Scheme 1.

As illustrated in the examples, in some embodiments of the invention theLNA nucleosides in the oligonucleotides are beta-D-oxy-LNA nucleosides.

Gapmer

The term “gapmer” as used herein refers to an antisense oligonucleotidewhich comprises a region of RNase H recruiting oligonucleotides (gap)which is flanked 5′ and 3′ by regions which comprise one or moreaffinity enhancing modified nucleosides (flanks or wings). Variousgapmer designs are described herein. Headmers and tailmers areoligonucleotides capable of recruiting RNase H where one of the flanksis missing, i.e. only one of the ends of the oligonucleotide comprisesaffinity enhancing modified nucleosides. For headmers the 3′ flank ismissing (i.e. the 5′ flank comprises affinity enhancing modifiednucleosides) and for tailmers the 5′ flank is missing (i.e. the 3′ flankcomprises affinity enhancing modified nucleosides).

Gapmer Designs

Gapmer oligonucleotides are widely used to inhibit a target RNA in acell, such as a mRNA or viral RNA, via an antisense mechanism (and maytherefore also be called antisense gapmer oligonucleotides). In a gapmerstructure the oligonucleotide comprises at least three distinctstructural regions a 5′-flank, a gap and a 3′-flank, F-G-F′ in '5->3′orientation. The G region or gap region comprises a region of at least 5contiguous nucleotides which are capable or recruiting RNaseH, such as aregion of DNA nucleotides, e.g. 6-14 DNA nucleotides or othernucleosides which are capable of recruiting RNase H, e.g.,alpha-L-oxy-LNA, 2′-Flouro-ANA and UNA. The gap region is flanked 5′ and3′ by regions (F and F′ also termed flanking regions or wing regions)which comprise one or more affinity enhancing modified nucleosides, suchas 2′ modified nucleotides. In some embodiments, the flanking regionsmay be 1-8 nucleotides in length.

In further embodiments region F (5′ flank or 5′ wing) is attached to the'5 end of region G and region F′ (3′ flank or 3′ wing) is attached tothe '3 end of region G. Region F and F′, comprises contains or consistsindependently of at least one modified nucleoside such as at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7 modifiednucleosides. In an embodiment region F and/or F′ comprises or consistsidependently of from 1 to 7 modified nucleosides, such as from 2 to 6modified nucleosides, such as from 2 to 5 modified nucleosides, such asfrom 2 to 4 modified nucleosides, such as from 1 to 3 modifiednucleosides, such as 1, 2, 3 or 4 modified nucleosides. The F region isdefined by having at least on modified nucleoside at the 5′ end and atthe 3′ end of the region. The F′ region is defined by having at least onmodified nucleoside at the 5′ end and at the 3′ end of the region.

In some embodiments, the modified nucleosides in region F and/or F′ havea 3′ endo structure.

In an embodiment, one or more of the modified nucleosides in region Fand/or F′ are 2′ modified nucleosides. In one embodiment all thenucleosides in Region F and/or F′ are 2′ modified nucleosides.

In another embodiment region F and/or F′ comprises DNA and/or RNA inaddition to the 2′ modified nucleosides. Flanks comprising DNA and/orRNA are characterized by having a 2′ modified nucleoside in the 5′ endand the 3′end of the F and/or F′ region. In one embodiment the region Fand/or F′ comprises DNA nucleosides, such as from 1 to 3 contiguous DNAnucleosides, such as 1 to 3 or 1 to 2 contiguous DNA nucleosides. TheDNA nucleosides in the flanks should preferably not be able to recruitRNase H. In some embodiments the 2′ modified nucleosides and DNA and/orRNA nucleosides in the F and/or F′ region alternate with 1 to 3 2′modified nucleosides and 1 to 3 DNA and/or RNA nucleosides. Such flankscan also be termed alternating flanks. The length of region F and/or F′in oligonucleotides with alternating flanks may independently be 4 to 10nucleosides, such as 4 to 8, such as 4 to 6 nucleosides, such as 4, 5, 6or 7 modified nucleosides. In some embodiments only the F region of theoligonucleotide is alternating. In some embodiments only the F′ regionof the oligonucleotide is alternating.

Specific examples of region F and/or F′ with alternating nucleosides are

-   -   2′₁₋₃-N′₁₋₄-2′₁₋₃    -   2′₁₋₂-N′₁₋₂-2′₁₋₂-N′₁₋₂-2′₁₋₂

Where 2′ indicates a modified nucleoside and N′ is a RNA or DNA. In someembodiments all the modified nucleosides in the alternating flanks areLNA and the N′ is DNA. In a further embodiment one or more of the 2′modified nucleosides in region F and/or F′ are independently selectedfrom 2′-O-alkyl-RNA units, 2′-O-methyl-RNA, 2′-amino-DNA units,2′-fluoro-DNA units, 2′-alkoxy-RNA, MOE units, LNA units, arabinonucleic acid (ANA) units and 2′-fluoro-ANA units.

In some embodiments the F and/or F′ region comprises both LNA and a 2′substituted modified nucleoside. These are often termed mixed wing ormixed flank oligonucleotides.

In one embodiment of the invention all the modified nucleosides inregion F and/or F′ are LNA nucleosides. In a further embodiment all thenucleosides in Region F and/or F′ are LNA nucleosides. In a furtherembodiment the LNA nucleosides in region F and/or F′ are independentlyselected from the group consisting of oxy-LNA, thio-LNA, amino-LNA, cET,and/or ENA, in either the beta-D or alpha-L configurations orcombinations thereof. In a preferred embodiment region F and/or F′comprise at least 1 beta-D-oxy LNA unit, at the 5′ end of the contiguoussequence.

In further embodiments, Region G (gap region) preferably comprise,contain or consist of at least 4, such as at least 5, such as at least6, at least 7, at least 8, at least 9, at least 10, at least 11, atleast 12, at least 13, at least 14, at least 15 or at least 16consecutive nucleosides capable of recruiting the aforementionednuclease, in particular RNaseH. In a further embodiment region Gcomprise, contain or consist of from 5 to 12, or from 6 to 10 or from 7to 9, such as 8 consecutive nucleotide units capable of recruitingaforementioned nuclease.

The nucleoside units in region G, which are capable of recruitingnuclease are in an embodiment selected from the group consisting of DNA,alpha-L-LNA, C4′ alkylated DNA (as described in PCT/EP2009/050349 andVester et al., Bioorg. Med. Chem. Lett. 18 (2008) 2296-2300, bothincorporated herein by reference), arabinose derived nucleosides likeANA and 2′F-ANA (Mangos et al. 2003 J. AM. CHEM. SOC. 125, 654-661), UNA(unlocked nucleic acid) (as described in Fluiter et al., Mol. Biosyst.,2009, 10, 1039 incorporated herein by reference). UNA is unlockednucleic acid, typically where the bond between C2 and C3 of the ribosehas been removed, forming an unlocked “sugar” residue.

In a still further embodiment at least one nucleoside unit in region Gis a DNA nucleoside unit, such as from 1 to 12 DNA units, such as 2, 3,4, 5, 6, 7, 8, 9, 10 or 11 DNA units, preferably from 2 to 12 DNA units,such as from 4 to 12 DNA units, more preferably from 5 to 11, or from 2to 10, 4 to 10 or 6 to 10 DNA units, such as from 7 to 10 DNA units,most preferably 8, 9 or 10 DNA units. In some embodiments, region Gconsists of 100% DNA units.

In further embodiments the region G may consist of a mixture of DNA andother nucleosides capable of mediating RNase H cleavage. Region G mayconsist of at least 50% DNA, more preferably 60%, 70% or 80% DNA, andeven more preferred 90% or 95% DNA.

In a still further embodiment at least one nucleoside unit in region Gis an alpha-L-LNA nucleoside unit, such as at least one alpha-L-LNA,such as 2, 3, 4, 5, 6, 7, 8 or 9 alpha-L-LNA.

In a further embodiment, region G comprises the least one alpha-L-LNA isalpha-L-oxy-LNA. In a further embodiment region G comprises acombination of DNA and alpha-L-LNA nucleoside units.

In some embodiments the size of the contiguous sequence in region G maybe longer, such as 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleosideunits.

In some embodiments, nucleosides in region G have a 2′ endo structure.

In the gapmer designs reported herein the gap region (Y′) may compriseone or more stereodefined phosphorothaiote linkage, and the remaininginternucleoside linkages of the gap region may e.g. be non-stereodefinedinternucleoside linkages, or may also be stereodefined phosphorothioatelinkages.

LNA Gapmer

The term LNA gapmer is a gapmer oligonucleotide wherein at least one ofthe affinity enhancing modified nucleosides is an LNA nucleoside. Insome embodiments both flanks of the gapmer oligonucleotide comprise atleast one LNA unit, and in some embodiments, all of the nucleoside ofthe flanks are LNA nucleosides.

In some embodiments, the 3′ flank comprises at least one LNA nucleoside,preferably at least 2 LNA nucleosides. In some embodiments, the 5′ flankcomprises at least one LNA nucleoside. In some embodiments both the 5′and 3′ flanking regions comprise a LNA nucleoside. In some embodimentsall the nucleosides in the flanking regions are LNA nucleosides.Typically the LNA load of the flanks of LNA gapmers is lower than thatfor 2′substituted nucleosides, and examples of LNA gapmer designsinclude [LNA]₁₋₄-[DNA]₅₋₁₅-[LNA]₁₋₄.

In some embodiments, the gapmer is a so-called shortmer as described inWO2008/113832 incorporated herein by reference.

Further gapmer designs are disclosed in WO2004/046160, WO2007/146511 andincorporated by reference.

Mixed Wing Gapmer

The term mixed wing gapmer or mixed flank gapmer refers to a LNA gapmerwherein at least one of the flank regions comprise at least one LNAnucleoside and at least one non-LNA modified nucleoside, such as atleast one DNA nucleoside or at least one 2′ substituted modifiednucleoside, such as, for example, 2′-O-alkyl-RNA, 2′-O-methyl-RNA,2′-alkoxy-RNA, 2′-O-methoxyethyl-RNA (MOE), 2′-amino-DNA, 2′-Fluoro-RNAand 2′-F-ANA nucleoside(s). In some embodiments the mixed wing gapmerhas one flank which comprises only LNA nucleosides (e.g. 5′ or 3′) andthe other flank (3′ or 5′ respectfully) comprises 2′ substitutedmodified nucleoside(s) and optionally LNA nucleosides.

Gapbreaker

The term “gapbreaker oligonucleotide” is used in relation to a gapmercapable of maintaining RNAseH recruitment even though the gap region isdisrupted by a non-RNaseH recruiting nucleoside (a gap-breakernucleoside, E) such that the gap region comprise less than 5 consecutiveDNA nucleosides. Non-RNaseH recruiting nucleosides are for examplenucleosides in the 3′ endo conformation, such as LNA's where the bridgebetween C2′ and C4′ of the ribose sugar ring of a nucleoside is in thebeta conformation, such as beta-D-oxy LNA or ScET nucleoside. Theability of gapbreaker oligonucleotide to recruit RNaseH is typicallysequence or even compound specific—see Rukov et al. 2015 Nucl. AcidsRes. Vol. 43 pp. 8476-8487, which discloses “gapbreaker”oligonucleotides which recruit RNaseH which in some instances provide amore specific cleavage of the target RNA.

In some embodiments, the oligonucleotide of the invention is agapbreaker oligonucleotide. In some embodiments the gapbreakeroligonucleotide comprise a 5′-flank (F), a gap (G) and a 3′-flank (F′),wherein the gap is disrupted by a non-RNaseH recruiting nucleoside (agap-breaker nucleoside, E) such that the gap contain at least 3 or 4consecutive DNA nucleosides. The gap-breaker design is based upon thegapmer designs, e.g. those disclosed here (e.g. Region F corresponds tothe X′ region of the gapmer above, and region F′ corresponds to theregion Z′ of the gapmer described herein), with the exception that thegap region (region Y′) comprises a gap-breaker nucleoside. In someembodiments the gapbreaker nucleoside (E) is an LNA nucleoside where thebridge between C2′ and C4′ of the ribose sugar ring of a nucleoside isin the beta conformation and is placed within the gap region such thatthe gap-breaker LNA nucleoside is flanked 5′ and 3′ by at least 3 (5′)and 3 (3′) or at least 3 (5′) and 4 (3′) or at least 4(5′) and 3(3′) DNAnucleosides, and wherein the oligonucleotide is capable of recruitingRNaseH.

The gapbreaker oligonucleotide can be represented by the followingformulae:

F-G-E-G-F′; in particular F₁₋₇-G₃₋₄-E₁-G₃₋₄-F′₁₋₇

D′-F-G-F′, in particular D′₁₋₃-F₁₋₇-G₃₋₄-E₁-G₃₋₄-F′₁₋₇

F-G-F′-D″, in particular F₁₋₇-G₃₋₄-E₁-G₃₋₄-F′₁₋₇-D″₁₋₃

D′-F-G-F′-D″, in particular D′₁₋₃-F₁₋₇-G₃₋₄-E₁-G₃₋₄-F′₁₋₇-D″₁₋₃

Where G represents DNA nucleosides and region D′ and D″ are optional andmay additional 5′ and/or 3′ nucleosides, such as DNA nucleosides.

In some embodiments the gapbreaker nucleoside (E) is a LNA, beta-D-oxyLNA or ScET or another LNA nucleoside, such as beta-D-nucleosidedisclosed herein.

Stereodefined Gapmers

In some embodiments, the child oligonucleotides originating from aparent gapmer oligonucleotide has at least one of the internucleosidelinkages of the gap region which is stereodefined, and optionallywherein the gap region comprises both Rp and Sp internucleosidelinkages.

In some embodiments, in the child oligonucleotide(s) (and optionally theparent), at least one of the internucleoside linkages of the gap regionare stereodefined, and wherein the central region comprises both Rp andSp internucleoside linkages.

In some embodiments, in the child oligonucleotide(s) (and optionally theparent), the internucleoside linkages within region G are allstereodefined phosphorothioate internucleoside linkages. In someembodiments, in the child oligonucleotide(s) (and optionally theparent), the internucleoside linkages within region F and F′ arestereodefined phosphorothioate internucleoside linkages. In someembodiments in the child oligonucleotide(s) (and optionally the parent),the internucleoside linkages between region F and G and between region Gand F′ are stereodefined phosphorothioate internucleoside linkages. Insome embodiments in the child oligonucleotide(s) (and optionally theparent), all the internucleoside linkages within the contiguousnucleosides of regions F-G-F′ are stereodefined phosphorothioateinternucleoside linkages.

The introduction of at least one stereodefined phosphorothioate linkagesin the gap region of an oligonucleotide may be used to modulate thebiological profile of the oligonucleotide, for example it may modulatethe toxicity profile. In some embodiments, 2, 3, 4 or 5 of thephosphorothioate linkages in the gap region in the childoligonucleotide(s) (and optionally the parent), are stereodefined. Insome embodiments the remaining internucleoside linkages in the gapregion are not stereodefined: They exist as a racemic mixture of Rp andSp in the population of oligonucleotide species. In some embodiments inthe child oligonucleotide(s) (and optionally the parent), the remaininginternucleoside linkage in the oligonucleotide are not stereodefined. Insome embodiments in the child oligonucleotide(s) (and optionally theparent), all the internucleoside linkages in the gap region arestereodefined.

In some embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 ofthe linkages in the gap region of the oligomer are stereoselectivephosphorothioate linkages.

In some embodiments 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the linkages in theoligomer (e.g. gapmer) are stereoselective phosphorothioate linkages. Insome embodiments all of the phosphorothioate linkages in the oligomerare stereoselective phosphorothioate linkages. In some embodiments theall the internucleoside linkages of the oligomer are stereodefinedphosphorothioate linkages.

RNase H Activity and Recruitment

The RNase H activity of an antisense oligonucleotide refers to itsability to recruit RNase H when in a duplex with a complementary RNAmolecule. WO01/23613 provides in vitro methods for determining RNaseHactivity, which may be used to determine the ability to recruit RNaseH.Typically an oligonucleotide is deemed capable of recruiting RNase H ifit, when provided with a complementary target nucleic acid sequence, hasan initial rate, as measured in pmol/l/min, of at least 5%, such as atleast 10% or more than 20% of the of the initial rate determined whenusing a oligonucleotide having the same base sequence as the modifiedoligonucleotide being tested, but containing only DNA monomers withphosphorothioate linkages between all monomers in the oligonucleotide,and using the methodology provided by Example 91-95 of WO01/23613(hereby incorporated by reference).

Efficacy and Therapeutic Index

The nucleic acid molecules identified by the method of the invention mayin addition the toxicity be tested as part of the method of theinvention to determine that they are still effective nucleic acidmolecules. In particular with nucleic acid based nucleic acid moleculesit has been observed that when toxicity is reduced this is in part dueto a reduction in the efficacy of the nucleic acid molecule. In oneaspect of the invention the decrease in the toxicity grade does notresult in a significant decrease in the efficacy. In some embodimentsthe therapeutic index (TI) of the nucleic acid molecule selectedaccording to the method of the invention is improved when compared to atoxic reference substance. For the purpose of the present invention TIrefers to the dose of the nucleic acid molecule that causes adverseeffects in the kidney in 50% of subjects (TD50) divided by the dose thatleads to the desired efficacy (target knockdown or pharmacologicaleffect) in 50% of subjects (EC50). In some embodiments the TI isincreased by at least 10%, such as at least 15, 25, 50, 75, 100, 150,200, 250 or 300% when compared to a toxic reference nucleic acidmolecule, such as Compound 4-1 herein or a toxic parent oligonucleotideor toxic parent nucleic acid molecule. For experimental purposes,assuming the target is present in mice, TD50 and EC50 may be determinedin mice in a seven day mouse study. If for example for nucleic acidmolecules, the sequence conservation in mice is unfavorable, other modelspecies may be used, e.g. rat, monkey, dog, pig or monkey (e.g.cynomolgus monkey). In some embodiments the increase in TI is due to anincreased TD50 and a low or no decrease in EC50. The increase in TI mayalso be the result of improvements on both parameters. In relation tothe method of the invention the increase in TI is not brought about byonly improving the efficacy of the nucleic acid molecule (i.e. reducedEC50).

The method of the invention may therefore comprise an additional step ofscreening the library of (e.g. child) nucleic acid molecules for theirefficacy in modulating, e.g. inhibiting, their target.

Alternatively, the method of the invention may comprise an additionalstep of testing the selected nucleic acid molecules (e.g. stereodefinedoligonucleotide variants) with a reduced toxicity to determine theirefficacy as nucleic acid molecules, e.g. as antisense oligonucleotides.

For antisense oligonucleotides the efficacy may be determined by theoligonucleotides ability to recruit RNaseH, or in some embodiments maybe the ability to modulate the expression of the target in a cell, invitro, or in some embodiments, in vivo.

Conjugate Moieties

In some embodiments, the conjugate moiety comprises or is acarbohydrate, non nucleosidic sugars, carbohydrate complexes. In someembodiments, the carbohydrate is selected from the group consisting ofgalactose, lactose, n-acetylgalactosamine, mannose, andmannose-6-phosphate.

In some embodiments, the conjugate moiety comprises or is selected fromthe group of protein, glycoproteins, polypeptides, peptides, antibodies,enzymes, and antibody fragments,

In some embodiments, the conjugate moiety is a lipophilic moiety such asa moiety selected from the group consisting of lipids, phospholipids,fatty acids, and sterols.

In some embodiments, the conjugate moiety is selected from the groupconsisting of small molecules drugs, toxins, reporter molecules, andreceptor ligands.

In some embodiments, the conjugate moiety is a polymer, such aspolyethyleneglycol (PEG), polypropylene glycol.

In some embodiments the conjugate moiety is or comprises aasialoglycoprotein receptor targeting moiety, which may include, forexample galactose, galactosamine, N-formyl-galactosamine,Nacetylgalactosamine, N-propionyl-galactosamine,N-n-butanoyl-galactosamine, and N-isobutanoylgalactos-amine. In someembodiments the conjugate moiety comprises a galactose cluster, such asN-acetylgalactosamine trimer. In some embodiments, the conjugate moietycomprises a GalNAc (N-acetylgalactosamine), such as a mono-valent,di-valent, tri-valent of tetra-valent GalNAc. Trivalent GalNAcconjugates may be used to target the compound to the liver (see e.g.U.S. Pat. No. 5,994,517 and Hangeland et al., Bioconjug Chem. 1995November-December; 6(6):695-701, WO2009/126933, WO2012/089352,WO2012/083046, WO2014/118267, WO2014/179620, & WO2014/179445), seespecific examples in FIG. 2 . These GalNAc references and the specificconjugates used therein are hereby incorporated by reference.

DETAILED DESCRIPTION OF THE INVENTION

Prediction of In Vivo Toxicity

The methods described herein may be used to predict the in vivo toxicityof a nucleic acid molecule in a mammal. Toxicity of any compound istypically dependent upon its dose, and as such the methods of theinvention may be used to assess a compounds comparative toxicity profileas compared to a negative control, and/or a nucleic acid molecule whosetoxicity profile is known, or compared to a population (or a library) ofnucleic acid molecules, such as a library of nucleic acid molecules, inparticular a library of antisense oligonucleotides. In this respect, theprediction of in vivo toxicity may be an assessment of the comparativerisk of encountering a toxicity phenotype, such as nephrotoxicity, whenthe nucleic acid molecule(s) are administered in vivo in a mammal.

When assessing the safety of a nucleic acid molecule, in particular anantisense oligonucleotide, one of the parameters that is investigated isthe impact of the substance on the kidney, e.g. accumulation in renalproximal tubules, tubular degenerative/regenerative changes(tubulotoxicity), biomarkers for acute kidney injury, like KIM-1, whichare detectable in the urine. Kidney related toxicities are generallyknown as nephrotoxicity.

The methods of the invention may be used to predict the in vivo toxicity(e.g. nephrotoxicity), or alternatively stated to determine the likelyin vivo toxicity profile (e.g. nephrotoxicity), of a nucleic acidmolecule in vivo in a mammal. In one embodiment of the invention the invivo nephrotoxicity is acute kidney injury and/or tubular degradation.The methods of the invention may be used to identify nucleic acidmolecules which are not toxic (e.g. not nephrotoxic) in vivo, when usedat dosages which are effective in modulating their target, or attherapeutically effective doses. The methods of the present inventiontherefore allows the selection of nucleic acid molecules which do notexhibit dose limiting toxicity (e.g. nephrotoxicity) when used in vivoat effective dosages. It will be recognized that it is an advantage tohave a wide safety margin when selecting nucleic acid molecules for invivo or for therapeutic use, and as such the methods of the inventionmay be used to identify or select nucleic acid molecules which do notelicit in vivo toxicity, such as nephrotoxicity, when dosed effectively,or at higher doses, e.g. at up to 2× the effective dose, or up to 3× theeffective dose, or up to 5× the effective dose, or up to 10× theeffective dose. The methods of the invention may therefore be used toidentify nucleic acid molecules for in vivo use which have a maximumtolerated dose higher (e.g. at least 2×, at least 3×, at least 5×, atleast 10×) than the effective dose. In this regard the methods of theinvention may be used to select nucleic acid molecules which have asuitable therapeutic index (TI) for safe therapeutic administration, oran improved therapeutic index as defined herein.

The present invention establishes EGFR as a reliable biomarker in an invitro cell based assay for prediction of in vivo nephrotoxicity of anucleic acid molecule.

One aspect of the present invention is an in vitro method for predictingin vivo nephrotoxicity of a nucleic acid molecule in a mammal, saidmethod comprising the steps of:

-   -   a) culturing cells expressing epidermal growth factor receptor        (EGFR) in a suitable cell culture media;    -   b) administering the nucleic acid molecule to said cell culture;    -   c) incubating the cells for a period of time; and    -   d) subsequently measuring the EGFR mRNA level in the cells;

wherein a decrease in EGFR mRNA in the cells is indicative of a nucleicacid molecule which is, or is predicted to be, associated withnephrotoxicity.

The decrease in intracellular EGFR mRNA levels can be evaluated inrelation to a reference value (control) obtained from non-treated cells,cells treated with vehicle (vehicle control, i.e the solvent in whichthe nucleic acid molecule is dissolved, e.g. saline, DMSO or othersuitable solvents) or cells treated with non-toxic reference nucleicacid molecule where the non-toxic nucleic acid molecule has beenvalidated as non-toxic in vivo (an in vivo validated non-toxic nucleicacid molecule). Such a non-toxic nucleic acid molecule can for examplebe antisense oligonucleotide compound 1-1 with SEQ ID NO: 1, inparticular if the nucleic acid molecule(s) that is being subjected tothe assay of the invention is a nucleic acid molecule(s), such as anantisense oligonucleotide(s).

In some embodiments, the alteration in the EGFR biomarker is determinedas the percentage of the reference value. As is illustrated in theexamples, the present inventors have found that a decrease in the levelof EGFR mRNA in the cells (intra cellular EGFR) below 80%, such as below75%, such as below 70%, such as below 60%, such as below 50%, such asbelow 40% relative to the reference value is predicative ofnephrotoxicity of the drug compound. In embodiments where the nucleicacid molecule is an antisense oligonucleotide the oligonucleotide isadministered to the cells in a concentration above 10 micro molar, suchas between 20 and 150 micro molar, such as between 30 and 120 micromolar, such as between 50 and 100 micro molar.

In further embodiments the EGFR mRNA level in the cells is furthercompared to a second reference value obtained from cells treated with anephrotoxic nucleic acid molecule, where the nephrotoxic referencenucleic acid molecule has been validated to cause nephrotoxicity in vivo(an in vivo validated nephrotoxic reference nucleic acid molecule). Insome embodiments, the method of the invention may therefore furthercomprise the method steps of the invention using the administration ofone or more nucleic acid molecules with a known toxicity (e.g.nephrotoxicity) profile, such as a positive control nucleic acidmolecule which is known to elicit in vivo nephrotoxicity and/or anegative control nucleic acid molecule which is known not to elicit invivo nephrotoxicity, and a comparison of the level of EGFR mRNA in thecells from the administration of the nucleic acid molecule(s) with thelevels obtained from the positive and/or negative controls. Thegeneration of data from of a negative control (vehicle control or invivo validated non-toxic nucleic acid molecule) and a positive control(in vivo validated nephrotoxic nucleic acid molecule) allowsdetermination of an assay window that can be used to normalize for batchto batch variation in the assay. The assay window (AW) is the differencebetween the non-toxic reference nucleic acid molecule and thenephrotoxic reference nucleic acid molecule.

In one embodiment the decrease in the level of EGFR mRNA is assed usingthe conditions for oligonucleotide treatment described in the “materialsand methods” section where the oligonucleotide is administered to thecells in a concentration above 1 micro molar, such as above 5 micromolar, such as above 10 micro molar, such as between 20 and 150 micromolar, such as between 30 and 120 micro molar, such as between 50 and100 micro molar.

In some embodiment the in vivo validated nephrotoxic reference nucleicacid molecule is antisense oligonucleotide compound 4-1 with SEQ ID NO:4.

In addition or alternatively, the control data may be determined bycomparing a library of nucleic acid molecules, in particular a libraryof nucleic acid molecules such as antisense oligonucleotides, RNAiagents or aptamers, using the method of the invention, either in seriesor in parallel, and comparing the level of EGFR mRNA biomarker afteradministration of each member of the library of nucleic acid molecules.Such a method allows for the selection of comparatively less toxic (suchas nephrotoxic) nucleic acid molecules. In some embodiments, the controldata may be or may include control data which is from a cell culturesample which has not been administered a nucleic acid molecule orvehicle control (day 0 control). Such a sample may be obtainedimmediately prior to the administration step.

Complementary Biomarkers

As is illustrated in the examples, the present inventors have found thatextracellular kidney injury molecule-1 (KIM-1) protein levels orintracellular KIM-1mRNA levels can serve as complementary biomarkers tothe EGFR biomarker further strengthening the predictability of themethod of the invention.

In some examples the EGFR biomarker alone was not sufficient to predictthe nephrotoxicity of a compound with known in vivo nephrotoxicity. Inthese instances KIM-1 as an in vitro biomarker showed to complement theEGFR biomarker very well. As illustrated in FIG. 2 the combination ofEGFR and KIM-1 biomarkers in the assay of the invention resulted in a100% prediction of compounds with known in vivo nephrotoxicity.

In an embodiment of the invention the method for predicting in vivonephrotoxicity of a nucleic acid molecule in a mammal, said methodcomprising the steps of:

-   -   a) culturing cells expressing epidermal growth factor receptor        (EGFR) in a suitable cell culture media;    -   b) administering the nucleic acid molecule to said cell culture;    -   c) incubating the cells for a period of time; and    -   d) subsequently measuring the EGFR mRNA level in the cells and        the KIM-1 protein levels in the supernatant;

wherein a decrease in the EGFR mRNA level or an increase in the KIM-1level in the supernatant is indicative of a nucleic acid molecule whichis, or is predicted to be, associated with nephrotoxicity.

As an alternative or supplement to the extracellular KIM-1 proteinlevels the intracellular level of KIM-1 mRNA may also be measured.

In some embodiments, the alteration in the KIM-1 protein biomarker isdetermined as the percentage of the reference value (control) obtainedfrom non-treated cells, cells treated with vehicle (vehicle control, i.ethe solvent in which the nucleic acid molecule is dissolved, e.g.saline, DMSO or other suitable solvents) or cells treated with non-toxicreference nucleic acid molecule where the non-toxic nucleic acidmolecule has been validated as non-toxic in vivo (an in vivo validatednon-toxic nucleic acid molecule). As is illustrated in the examples, thepresent inventors have found that an increase in the level of KIM-1 inthe supernatant (extracellular KIM-1) above 175%, such as at least 200%,such as at least 250%, such as at least 300%, such as at least 350%,such as at least 400% relative to the reference value is predicative ofnephrotoxicity of the drug compound. In some embodiments, the alterationin the intracellular KIM-mRNA biomarker is determined as the percentageof the control reference value. As is illustrated in the examples, thepresent inventors have found that an increase in the level of KIM-1 mRNAin the cells (intracellular KIM-1) above 500%, such as at least 750%,such as at least 1000%, such as at least 1100%, such as at least 1300%,such as at least 1500%, such as at least 1700%, such as at least 2000%relative to the reference value is predicative of nephrotoxicity of thedrug compound, An increase in KIM-1 levels is predictive even if therefor the same compound is no observable decrease in EGFR levels. Withoutbeing bound by theory we suspect that the mechanism that elicits theKIM-1 biomarker increase in vitro is different from the mechanism thatelicits the increase in extracellular EGFR levels.

In addition to the EGFR and KIM-1 biomarkers, the inventors haveidentified an additional biomarkers that can supplement the EGFRbiomarker if the cell culture media comprise at least 4 ng/ml ofepidermal growth factor (EGF). The additional biomarker is intracellularadenosine triphosphate (ATP).

In a further aspect of the invention ATP can be used as a supplementalbiomarker to EGFR in an in vitro cell based assay for prediction of invivo nephrotoxicity of a nucleic acid molecule.

In another embodiment of the invention the method for predicting in vivonephrotoxicity of a nucleic acid molecule in a mammal, said methodcomprising the steps of:

-   -   a) culturing cells expressing epidermal growth factor receptor        (EGFR) in a suitable cell culture media containing at least 4        ng/ml of epidermal growth factor (EGFR);    -   b) administering the nucleic acid molecule to said cell culture;    -   c) incubating the cells for a period of time; and    -   d) subsequently measuring the intracellular EGFR mRNA level and        the intracellular ATP levels;

wherein an decrease in the EGFR mRNA level in the cells and a decreasein the intracellular ATP protein level is indicative of a nucleic acidmolecule which is, or is predicted to be, associated withnephrotoxicity.

A decrease of cellular ATP in relation to the control to at least 80%,such at least about 70%, such as at least about 60%, such as at least50%, such as at least 40% relative to the reference value is indicativeof an enhanced propensity to trigger nephrotoxicity in vivo. The ATPbiomarker is compared to a set of controls as already described inrelation to the EGFR biomarker. In particular to a negative controlreference value obtained from non-treated cells, cells treated withvehicle (vehicle control, i.e the solvent in which the nucleic acidmolecule is dissolved, e.g. saline, DMSO or other suitable solvents) orcells treated with non-toxic reference nucleic acid molecule where thenon-toxic nucleic acid molecule has been validated as non-toxic in vivo(an in vivo validated non-toxic nucleic acid molecule). Such a non-toxicnucleic acid molecule can for example be compound 1-1 with SEQ ID NO: 1,in particular if the nucleic acid molecule(s) that is being subjected tothe assay of the invention is a nucleic acid molecule(s), such as anantisense oligonucleotide(s). The intracellular ATP biomarker mayfurther be compared to a second reference value obtained from cellstreated with a nephrotoxic nucleic acid molecule, where the nephrotoxicreference nucleic acid molecule has been validated to causenephrotoxicity in vivo (an in vivo validated nephrotoxic referencenucleic acid molecule).

In embodiments the cell culture medium comprises between 3 and 20 ngEGF/ml culture medium, such as between 5 and 15 ng/ml, such as between 6and 10 ng/ml, such as at least 3 ng/ml, such as at least 4 ng/ml, suchas at least 5 ng/ml, such as at least 6 ng/ml, such as at least 7 ng/ml,such as at least 8 ng/ml, such as at least 9 ng/ml. In a preferredembodiment the cell culture medium comprises between 8 and 15 ng/ml. Ina further preferred embodiment the cell culture medium comprises atleast 10 ng EGF/ml culture medium. This is in particular relevant whereATP is used as supplementary biomarkers.

It should be recognized that the actual level of increase/decrease ofthe biomarker will depend on many factors including the properties ofthe cell culture, the density of the cell culture, the concentration ofnucleic acid molecule used, and the incubation time of the nucleic acidmolecules. Relevant parameters are described in the following sections.

Cell Cultures

In the examples of the present invention several cell cultures have beentested. Both primary cell cultures as well as immortalized cell culturesappear to function in the method of the invention as long as theyexpress epidermal growth factor receptor (EGFR). For the EGFR and KIM-1biomarkers the EGFR receptor does not necessarily need to be functional(i.e. facilitating the consumption of EGF in the medium by the cells). Afunctional EGF receptor is however needed if biomarkers like ATP are tobe used in the present assay. The EGFR is endogenous, meaning that it isexpressed by the cell.

The method of the invention may be used to determine the likely toxicityin vivo of the nucleic acid molecule(s) in model species such as rodentspecies such as mouse, rat or rabbit, or pig (e.g. minipig) or dog, orprimates, such as monkeys (e.g. cynomolgus monkey), or may be used todetermine the likely toxicity in vivo of the nucleic acid molecule(s) inhumans. The inventors have found that the use of primary EGFR expressingepithelial cells or primary hepatocytes or immortalized EGFR expressingepithelial cell are predictive of the toxicity profile seen in vivo onrodent studies as well as in human clinical trials. In particularepithelia cells which express EGFR and originate from kidney,gastrointestinal tract or lung tissue or primary hepatocytes aresuitable in the method of the invention. In one embodiment theepithelial cell does not originate from the eye, in particular not fromthe retinal pigment epithelia, in particular the cell culture is not anARPE19 cell culture.

In one embodiment of the invention the cells express functional EGFR, inthat the cells consume EGF present in the growth medium when culturedunder normal conditions (without being subjected to drug or vehiclesubstances). The rate of EGF consumption is preferably at least 50% EGFfrom the medium over 72 hours, when the cells are grown under regularconditions without being subjected to drug or vehicle substances. Somecell cultures may have a higher rate of EGF consumption such as at least50% of the EGF in the medium over 60 hours, such as at least 50% of theEGF in the medium over 48 hours. In order to measure the EGF consumptionin a cell culture it is important that the medium comprises EGF.

In one embodiment cells which express EGFR can be selected from thegroup consisting of epithelial cell, endothelial cell, mesenchymalcells, neuroectodermal cells and hepatocytes. In particular cellcultures originating from epithelial cells or hepatocytes are used inthe method of the present invention.

In one embodiment the epithelial cells can be in the form of a mammalianprimary cell culture, such as a cell culture selected from the groupconsisting of rodent primary epithelial cells, such as mouse or ratprimary epithelial cells; pig (e.g. minipig) epithelial cells, dogepithelia cells and non-human primate primary epithelial cells, such asmonkey (e.g. cynomolgus monkey) or human primary epithelial cells. Inanother embodiment the hepatocytes can be in the form of a mammalianprimary cell culture, such as a cell culture selected from the groupconsisting of rodent primary hepatocytes, such as mouse or rat primaryhepatocytes; pig (e.g. minipig) hepatocytes, dog epithelia cells andnon-human primate primary hepatocytes, such as monkey (e.g. cynomolgusmonkey) or human primary hepatocytes. In a preferred embodiment theprimary epithelial or hepatocyte cell culture is obtained from a rat ora human. In addition to being obtainable from various species the cellculture may also be obtained from various organs, such as epithelialcells from the gastrointestinal tract, lungs, reproductive and urinarytracts, kidney, exocrine and endocrine glands. In one embodiment theepithelial cell culture is a kidney epithelial cell culture,gastrointestinal tract epithelial cell culture or a lung epithelial cellculture. Since the present invention sets out to predict nephrotoxicitykidney epithelial cells from selected from the group consisting ofproximal tubule epithelial cells, distal tubule epithelial cells andcollecting duct epithelial cells are particularly relevant. Specificexamples of primary cell cultures are those made from human PTEC or ratPTEC cells. In addition to epithelial cell cultures the examples of thepresent invention also show that other cell types expressing EGFR can beused, for example primary hepatocytes.

In another embodiment the cells expressing EGFR are cultured from animmortalized cell line. Specific examples of cell cultures obtained fromimmortalized cell lines are human PTEC-TERT-1, ciPTEC, CACO2, HK-2,NKi-2 or human A549 cell lines. An example of an immortalized cell linewhich does express EGFR, but does not have EGFR consumption is CACO2cells.

Administering the Nucleic Acid Molecule

The nucleic acid molecule to be assessed in the method of the presentinvention can be administered to the cell culture by common methodsknown in the art, such as passive diffusion, electroporation,sonication, cell squeezing, hydrostatic pressure, nanoparticles (e.g.liposomes), magnetofection and cell penetrating peptides. Nucleic acidmolecules are generally administered using transfection or through theprocess known as gymnosis (also known as naked delivery, see Stein etal., NAR 2010 38(1) e3 or Soifer et al., Methods Mol Biol 2012, 815:333-46), although other administration options also can be applied. Inthe examples it can be seen that the prediction of the in vitronephrotoxicity using the EGFR biomarker is independent on whether theantisense oligonucleotide is administered to the cell culture bytransfection or by gymnosis. The mode of administration needs to beadapted to the properties of the cell culture. Some cell types are moreprone to take oligonucleotides up by gymnosis, as demonstrated for PTECtype cell lines, whereas other cell lines are more suitable fortransfection as seen for the A549 cell line. The mode of administrationis therefore subject to optimization such that the best mode ofadministration is investigated for a particular cell culture.

In one embodiment of the invention the nucleic acid molecule isadministered by gymnosis, in particular if the nucleic acid molecule issingle stranded oligonucleotides such as antisense oligonucleotides orsingled stranded RNAi agents. Since its discovery over 5 years ago,gymontic delivery has become a standard tool used in oligonucleotideresearch, and is a well-established term used in the art. Typically,gymnotic delivery of oligonucleotides utilizes a concentration ofoligonucleotide of between about 1 μM and about 1000 μM, such as betweenabout 5 μM and about 100 μM, such as between about 10 μM and about 50μM, such as between about 20 μM and 40 μM such as about 25-35 μM.Suitably oligonucleotides may be administered to the cell culture, e.g.in PBS, to achieve a final concentration of 1-100 μM, such as 5-50 μM,such as 10 or 30 μM. One of the advantages of gymnotic delivery is thatit resembles the delivery that occur in vivo, in that no additionalchemicals are needed to get the oligonucleotides into the cell.

In another embodiment of the invention the nucleic acid molecule isadministered by transfection, this approach is suitable both for singlestranded and double stranded oligonucleotides. Transfection can beperformed using standard methods known in the art, such as lipofection,cationic polymers, calcium phosphate etc. Typically, transfectionutilize nucleic acid molecule concentration of between 0.5 ng and 50 ng,such as between 1 ng and 25 ng, such as between 2 ng and 15 ng, such asbetween about 3 ng and 10 ng. Suitably oligonucleotides may beadministered to the cell culture, e.g. in PBS, to achieve a finalconcentration corresponding to those indicated above.

Incubation Time

The time between administration of the nucleic acid molecule and themeasurement of the relevant biomarkers is the incubation time orculturing time. For an in vitro toxicity assay to be as effective aspossible the readout needs to be reproducible and consistently predictcompounds with potential toxicity issues. Another relevant parameter isthe time it takes to conduct the assay, the quicker it can be done thehigher the throughput can be, which is highly relevant if the method ofthe invention is used to screen large libraries of nucleic acidmolecules. The incubation time is subject to optimization depending oncell culture and method of administration. In one embodiment of theinvention the incubation time with the nucleic acid molecule is between1 and 9 days such as between 2 and 8 days, 3 and 7 days, or between 4and 6 days, in particular between 2 and 6 days or between 3 and 6 days,such as 2, 3, 4, 5, 6, 7, 8 or 9 days. The inventors have found that forthe biomarkers, EGFR and Kim-1, a sufficient reproducibility can beachieved using a 2 to 6 day incubation time, in particular biomarkermeasurements at day 3 and day 6 after administration show good results.In one embodiment of the invention a single stranded oligonucleotide isadministered to the cell culture using gymnosis in a concentration of 50to 100 μM and incubated for 3 to 6 days and EGFR and/or KIM-1 ismeasured at these times.

In the case where the intracellular biomarker, ATP, is measured theincubation time the incubation time with the nucleic acid molecule isbetween 5 and 10 days, such as between 6 and 9, in, such as 6, 7, 8 or 9days.

In embodiments where the administration is performed using transfectionthe incubation time with the nucleic acid molecule is between 1 and 4days, such as between 2 and 3 days, such as 1, 2, 3 or 4 days. In oneembodiment of the invention a nucleic acid molecule is administered tothe cell culture using transfeciton in a concentration of 1 to 20 ng andincubated for 1 to 3 days and EGFR and/or KIM-1 is measured at thesetimes.

Cell culture of mammalian cells is typically performed at or about 37°C., and may further comprise exogenous CO₂, such kept in an atmosphereof or about 5% CO₂. Cells in culture generally needs fresh medium on aregular basis such as every 2 to 4 days. If the incubation time exceeds3 to 4 days the medium can be exchanged every 3 to 4 days. The freshmedium preferably contains a concentration of nucleic acid moleculecorresponding to the concentration given a day 0, to secure a continuespresence of the nucleic acid molecule during the incubation time.

One of the advantages of the present invention is the early read out ofpredictive toxicity biomarkers, relatively shortly after the initiationof the incubation period, and that the biomarkers used provide areliable signal.

Screening Library of Variants to Identify Child Nucleic Acid Moleculeswith a Predicted Reduced Toxicity Profile In Vivo The invention providesfor a method for selecting one or more nucleic acid molecules suitablefor in vivo administration to a mammal, from a library of nucleic acidmolecules, said method comprising the steps of:

-   -   a) Obtaining a library of nucleic acid molecules    -   b) Administer each member of the library of nucleic acid        molecules to a cell culture expressing epidermal growth factor        receptor (EGFR), such as described in the section “cell        cultures”;    -   c) and culturing the cells in vitro for a period of time such as        described in the section “incubation time”;    -   d) measuring the amount of at least one nephrotoxicity biomarker        such as described in the sections (Prediction of in vivo        toxicity” and “complementary biomarkers”; and    -   e) selecting one or more nucleic acid molecules wherein the %        reduction in EGFR relative to a reference value is above 80%,        such as above 85%, such as above 90, such as above 95%, such as        100% or above relative to reference value.

In one embodiment of the invention the therapeutic index of the nucleicacid molecule selected in step e) is decreased when compared to a toxicreference substance or parent nucleic acid molecule.

The selected nucleic acid molecule(s) may optionally administering invivo to the mammal to confirm that the compound does not elicitnephrotoxicity. In particular rats are highly sensitive to nephrotoxicnucleic acid molecules, and therefore a relevant species for confirmingthe prediction obtained using the method of the present invention.Naturally other mammalian species mentioned herein may also be relevant.

In some embodiments, the library of nucleic acid molecules is a libraryof antisense oligonucleotides.

In one embodiment the library of nucleic acid molecules have differentnucleobase sequences, for example they may be a library of nucleic acidmolecules which are designed across a target sequence (e.g. a mRNA), forexample a library of antisense oligonucleotides or RNAi agents generatedby a mRNA gene-walk.

In one embodiment the members of the library of nucleic acid moleculeshave identical nucleobase sequences in a contiguous stretch of thesequence, where the contiguous stretch is shorter than the nucleic acidmolecule, with at least a subset of the molecules in the library. Such a“hot-spot” library may for example be a library of nucleic acidmolecules which are designed across a target sequence of e.g 50 to 100nucleobases in length (e.g. a hot spot on a target RNA), where themembers of the library is will be overlapping along this sequence withat least 4 to 14 nucleobases, such as 5 to 12 nucleobases such as 6 to10 nucleobases. The library may for example be a library of antisenseoligonucleotides or RNAi agents identifying the oligonucleotide with thehighest TI targeting the identified hot-spot.

In some embodiments, the library of nucleic acid molecules is a libraryof nucleic acid molecule variants (child nucleic acid molecules) of aparent nucleic acid molecule, wherein the parent nucleic acid moleculeis toxic, such as nephrotoxic, and wherein step d) identifies one ormore nucleic acid molecule variants which are less toxic than the parentnucleic acid molecule; wherein the nucleic acid molecule variantsretaining the core nucleobase sequence of the parent nucleic acidmolecule, such as an antisense oligonucleotide.

In some embodiments, the child nucleic acid molecules may be the samelength as the parent nucleic acid molecule and retain the samenucleobase sequence. However, it is envisaged that, in some embodiments,the child nucleic acid molecules may be truncated, such as by theremoval of a 5′ and/or 3′ terminal nucleotide, or may in someembodiments, have an additional nucleotide at the 5′ and/or 3′ end.Removal of one or more terminal high affinity nucleosides, such as a LNAnucleoside allows for the affinity of the nucleic acid molecule to theRNA target to be maintained, as the insertion of one or more LNAnucleosides into the gap region will increase the affinity to the RNAtarget. It is envisaged that, in some embodiments, the library of childnucleic acid molecules may comprise variants which have different flankregions, some being truncated, some having additional nucleosides, somehaving a sequence shifted one or two nucleosides (as measured to the RNAtarget), some with additional high affinity nucleosides in the flanks,so the library is a complex library of stereodefined phosphorothioateoligonucleotides with heterogeneous phosphorothioate internucleosidelinkages, thereby allowing for the concurrent selection of child nucleicacid molecules which have a decreased toxicity as compared to the parentnucleic acid molecule.

The parent and child nucleic acid molecules share a common corenucleobase sequence. The common core nucleobase sequence (or contiguousnucleobase sequence) is typically at least 10 nucleobases long, such asat least 11, at least 12, at least 13, at least 14, at least 15, or atleast 16 nucleobases long, and in some embodiments may be the samenucleobase sequence of the parent nucleic acid molecule. In someembodiments the parent and (at least a proportion of) the child nucleicacid molecules have the same nucleobase sequence across the length ofthe nucleic acid molecules. It is however envisaged that a proportion ofthe child nucleic acid molecules may, in some embodiments, compriseadditional 5′ or 3′ nucleotides, such as an additional 1, 2 or 3 5′ or3′ nucleotides. In addition or alternatively in some embodiments, aproportion of the child nucleic acid molecules may be truncated withregards the parent, e.g. may comprise 1, 2 or 3nt truncation at the 5′or 3′ end. In some embodiments, additional nucleobase or truncations ofthe nucleobase sequence of the (proportion of) child nucleic acidmolecule (s) is a single nucleobase addition or truncation. In someembodiments, the child oligonucleotides, or a proportion thereof, may beshifted by a single nucleobase, or by 2 or 3 nucleobases in comparisonto the parent nucleic acid molecule when aligned to the target sequence(in effect a truncation at one end, and an addition at the other).Additional nucleotides retain complementarity with the target nucleicacid sequence.

In some embodiments the library of nucleic acid molecule variants (childnucleic acid molecules) differs from the parent nucleic acid molecule inone or more design parameters. The design parameter can be selected fromthe group consisting of i) presence of one or more stereodefinedphosphorothioate internucleoside linkages; ii) change in gap size; iii)introduction of a gap breaker; iv) change of wing size; v) introductionof 2′ sugar modified nucleosides; and vi) change in the 2′ sugarmodified nucleoside composition in the wings introducing at least twodifferent 2′ modified nucleosides in the wings, such as LNA nucleosidesand 2′ substituted nucleosides, in particular LNA and MOE. The designparamters ii), iii), iv) and vi) are in particular for antisenseoligonucleotides.

In some embodiments, the nucleic acid molecule variants are areantisense oligonucleotides.

In some embodiments the antisense oligonucleotide, is a gapmeroligonucleotide.

In some embodiments, the nucleic acid molecule variants are RNAi agents.

In some embodiments, the nucleic acid molecule variants differ from theparent nucleic acid molecule by the presence of one or morestereodefined phosphorothioate internucleoside linkages. Compounds 8-2and 8-3 as well as compounds 10-2 to 10-5 are examples of suchstereodefined variant molecules, where the parent molecules arecompounds 8-1 and 10-1 respectively.

In some embodiments, the antisense oligonucleotide variants are LNAoligonucleotides.

In some embodiments, the library of child antisense oligonucleotides areor comprise a population of child oligonucleotides with different gapmerdesigns, optionally including different mixed wing gapmer designs,gap-breaker designs, gap length and flank length.

The method of the invention may be used to identify stereodefinednucleic acid molecules with reduced in vivo toxicity (such asnephrotoxicity), in particular stereodefined antisense oligonucleotides.

The invention therefore provides for a particular method of reducing thetoxicity of an antisense oligonucleotide (parent oligonucleotide) usingstereodefined phosphorothioate internucleoside linkage, comprising thesteps of

-   -   a) Creating a library of stereodefined oligonucleotide variants        (child oligonucleotides), retaining the core nucleobase sequence        of the parent oligonucleotide;    -   b) Screening the library created in step a) in a cell culture        expressing epidermal growth factor receptor (EGFR); and    -   c) Identifying one or more stereodefined variants present in the        library which has a reduced toxicity in the cell culture as        compared to the parent oligonucleotide.

Optionally the method is repeated (reiterative screening), for exampleso that the one or more stereodefined variants identified by the methodis used as a parent oligonucleotide in the next round of the screeningmethod.

In the method of the invention, each member of the library created instep b) comprises at least one stereodefined phosphorothioateinternucleoside linkage which differs from parent.

The methods of the invention may further comprise an additionalsubsequent step of manufacturing the one or more selected nucleic acidmolecule variants which have a reduced toxicity using one of the methodsof the invention. In some embodiments, the subsequent manufacture is ina scale of more than 1 g, such as more than 10 g. In some embodiments,the synthesis of the oligonucleotides for in vivo or in vitro screeningsteps is performed at a scale of less than 1 g, such as less than 0.5 g,such as less than 0.1 g.

In some embodiments, the methods further comprise the step ofdetermining the in vitro or in vivo potency of either the library ofnucleic acid molecules, or of the one or more selected nucleic acidmolecules in the library identified in step c) or e).

The invention provides a method for predicting the (e.g. likely) in vivonephrotoxicity of an antisense oligonucleotide, such as a LNAoligonucleotide, said method comprising the steps of administering theoligonucleotide to a cell culture expressing epidermal growth factorreceptor (EGFR) optionally where the medium comprises at least 4 ng/mlof epidermal growth factor (EGF), incubating the cells in the presenceof the oligonucleotide, e.g. for a period of between 2-9 days, such as2-6 days, such as 3 days, and subsequently measuring at least onebiomarker of toxicity, such as those described herein, e.g. by measuringthe amount of EGFR mRNA, KIM-1 mRNA or ATP levels in the cell and/or theamount of KIM-1 released into the culture media. Suitably a reduction incellular EGFR mRNA levels or ATP levels is indicative of a nephrotoxicoligonucleotide, and elevation of KIM-1 in the culture media isindicative of a nephrotoxic oligonucleotide. In one embodiment the EGFRbiomarker is measured, optionally combined with measurement of KIM-1and/or ATP biomarkers.

The invention provides for the use of an in vitro assay to determine the(e.g. likely) neperotoxicity of a nucleic acid molecule, such as anucleic acid molecule, such as an antisense oligonucleotide, such as anLNA oligonucleotide.

It will be recognized that, in some embodiments, the methods forpredicting (or determining) the in vivo toxicity (e.g. nephrotoxicity),may be used to identify stereodefined variants of a parentoligonucleotide, where the sterodefined variants have reduced in vitroor in vivo toxicity.

Complementary Toxicity Assays

In addition to the method for predicting in vivo nephrotoxicity of adrug substance described in this application, the method may further becombined with methods for predicting other toxicities relevant to thedevelopment of a drug substance, in particular a therapeuticoligonucleotide, such as an antisense oligonucleotide.

In some embodiments, the method for predicting nephrotoxicity disclosedor claimed in the present application may be combined with a method forpredicting immunotoxicity of drug substance, in particular anoligonucleotide.

One method for predicting immunotoxicity of a drug substance inparticular of an oligonucleotide, comprises measuring at least onecomplement biomarker and/or at least one (such as at least two) cytokinebiomarkers in blood samples subjected to the drug substance.

In a particular embodiment immunotoxicity is predicted by measuring atleast one complement biomarker and at least two cytokine biomarkers inblood samples subjected to the drug substance, in particular anoligonucleotide.

In some embodiments the method of the invention further includes orprovides a method of predicting immunotoxicity comprising the steps ofa) administering the drug substance, in particular an oligonucleotide,to human blood (isolated from the body); b) incubating the samplesbetween 30 min to 8 hours; c) stop the reaction; and d) measure at leasttwo, three, four, five of or all of the following biomarkers: i)complement biomarkers C3a and C5a, and/or ii) cytokine biomarkersinterleukin 6 (IL6), interleukin 8 (IL8), tumor necrosis factor alpha(TNFa), and monocyte chemoattractant protein-1 (MCP1); wherein a meanincrease above about 2 fold compared to a control in at least two of thebiomarkers is indicative of in vivo immunotoxicity of the drugsubstance, such as the oligonucleotide. The blood sample of step a) istypically obtained from at least one healthy human subject, such as atleast two or at least three healthy human subjects. Generally, the bloodsamples are not mixed. In some embodiments at least one of thecomplement biomarkers C3a and C5a is measured in combination with atleast two cytokine biomarkers selected from the group consisting ofinterleukin 6 (IL6), interleukin 8 (IL8), tumor necrosis factor alpha(TNFa), and monocyte chemoattractant protein-1 (MCP1). In someembodiments at least cytokine biomarkers interleukin 8 (IL8), andmonocyte chemoattractant protein-1 (MCP1) are measured.

In some embodiments blood samples from at least 3 donors are used.Typically the blood is obtained from healthy donors (i.e. isolated fromthe body). In some embodiments the measurements from the 3 differentblood samples are averaged to achieve an immunotoxicity read out.

In some embodiments, the method disclosed or claimed in the presentapplication may be combined with a method for predicting hepatotoxicityof a drug substance, in particular an oligonucleotide. A method forpredicting in vivo hepatotoxicity of an oligonucleotide is described inWO2017/067970, hereby incorporated by reference. In brief one method ofpredicting in vivo hepatotoxicity of an oligonucleotide comprises thesteps of a) administering the oligonucleotide to a population of primarymammalian hepatocyte cells (or population of hepatocytes derived frominduced pluripotent stem cells) in vitro in a cell culture media; b)culturing the cells in vitro in the cell culture media for a period oftime, such as between 1 and 14 days, in particular between 2 and 7 days;and c) subsequently measuring the level of lactate dehydrogenase (LDH)released into the culture media, and/or measuring the level of cellularATP levels; wherein an increase in lactate dehydrogenase in the cellculture media, such as a 20% increase compared to a control, or adecrease in cellular ATP levels, such as a 20% decrease compared to acontrol, is indicative of an oligonucleotide which is, or is predictedto be hepatotoxic in vivo in a mammal. Further biomarkers that cansupplement the hepatotoxicity prediction are microRNA-122 released intothe culture media, where an increase in microRNA-122 in the cell culturemedium is predictive of hepatotoxicity, and intracellular glutathione(GSH) levels, where a reduction in GHS levels is predictive ofhepatotoxicity.

In some embodiments of the invention the method for predicting in vivonephrotoxicity is combined with a method of predicting in vivoimmunotoxicity and a method of predicting in vivo hepatotoxicity, suchas the methods described above.

Pharmaceutical Composition

In a further aspect, the invention provides a nucleic acid molecule orsalts thereof obtained by the method of the invention. In a furtheraspect the nucleic acid molecule or salts thereof obtained by the methodof the invention may be formulated into a pharmaceutical compositioncomprising any of the aforementioned nucleic acid molecule and apharmaceutically acceptable diluent, carrier, salt and/or adjuvant. Apharmaceutically acceptable diluent includes phosphate-buffered saline(PBS) and pharmaceutically acceptable salts include, but are not limitedto, sodium and potassium salts. In some embodiments the pharmaceuticallyacceptable diluent is sterile phosphate buffered saline. In someembodiments the oligonucleotide is used in the pharmaceuticallyacceptable diluent at a concentration of 50-300 μM solution.

Suitable formulations for use in the present invention are found inRemington's Pharmaceutical Sciences, Mack Publishing Company,Philadelphia, Pa., 17th ed., 1985. For a brief review of methods fordrug delivery, see, e.g., Langer (Science 249:1527-1533, 1990). WO2007/031091 provides further suitable and preferred examples ofpharmaceutically acceptable diluents, carriers and adjuvants (herebyincorporated by reference). Suitable dosages, formulations,administration routes, compositions, dosage forms, combinations withother therapeutic agents, pro-drug formulations are also provided inWO2007/031091.

Nucleic acid molecules obtained by the method of the invention may bemixed with pharmaceutically acceptable active or inert substances forthe preparation of pharmaceutical compositions or formulations.Compositions and methods for the formulation of pharmaceuticalcompositions are dependent upon a number of criteria, including, but notlimited to, route of administration, extent of disease, or dose to beadministered.

These compositions may be sterilized by conventional sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile aqueous carrier prior toadministration. The pH of the preparations typically will be between 3and 11, more preferably between 5 and 9 or between 6 and 8, and mostpreferably between 7 and 8, such as 7 to 7.5. The resulting compositionsin solid form may be packaged in multiple single dose units, eachcontaining a fixed amount of the above-mentioned agent or agents, suchas in a sealed package of tablets or capsules. The composition in solidform can also be packaged in a container for a flexible quantity, suchas in a squeezable tube designed for a topically applicable cream orointment.

The invention provides methods for treating or preventing a disease,comprising administering a therapeutically or prophylactically effectiveamount of a nucleic acid molecule or salts thereof obtained by themethod of the invention or a pharmaceutical composition of the inventionto a subject suffering from or susceptible to the disease.

The invention also relates to a nucleic acid molecule or salts thereofobtained by the method of the invention or a pharmaceutical compositionas defined herein for use as a medicament.

The nucleic acid molecule or salts thereof obtained by the method of theinvention or a pharmaceutical composition according to the invention istypically administered in an effective amount.

The invention also provides for the use of the nucleic acid molecule orsalts thereof obtained by the method of the invention for themanufacture of a medicament for the treatment of a disorder as referredto herein, or for a method of the treatment of as a disorder as referredto herein.

Embodiments of the Invention

The following embodiments of the present invention may be used incombination with any other embodiments described herein.

1. An in vitro method for predicting in vivo nephrotoxicity of a nucleicacid molecule in a mammal, said method comprising the steps of:

-   -   a. culturing cells expressing epidermal growth factor receptor        (EGFR) in a suitable cell culture media;    -   b. administering the nucleic acid molecule to said cell culture;    -   c. incubating the cells for a period of time; and    -   d. subsequently measuring the EGFR mRNA level in the cells;

wherein a decrease in EGFR mRNA levels is indicative of a nucleic acidmolecule which is, or is predicted to be, associated withnephrotoxicity.

2. The method according to embodiment 1, wherein EGFR mRNA level iscompared to a reference value obtained from cells treated with vehiclecontrol or a non-toxic reference nucleic acid molecule, where thenon-toxic reference nucleic acid molecule has been validated asnon-toxic in vivo.

3. The method according to embodiment 2, wherein the non-toxic referencenucleic acid molecule is an antisense oligonucleotide compoundconsisting of CGTcagtatgcgAATc (SEQ ID NO: 1), wherein lower caseletters represent DNA units, bold upper case letters representbeta-D-oxy-LNA units, all LNA C are 5′methyl C and all internucleosidelinkages are phosphorothioate linkage.

4. The method according to embodiment 3, wherein a level of EGFR mRNAbelow 80% relative to the vehicle control or non-toxic reference valueis predicative of nephrotoxicity of the nucleic acid molecule.

5. The method according to embodiment 2 to 4, wherein EGFR mRNA level isfurther compared to a second reference value obtained from cells treatedwith a nephrotoxic reference nucleic acid molecule, where thenephrotoxic reference nucleic acid molecule has been validated to causenephrotoxicity in vivo.

6. The method according to embodiment 5, wherein the toxic referencenucleic acid molecule is an antisense oligonucleotide compoundconsisting of GCtgtgtgagcttGG (SEQ ID NO: 4), wherein lower case lettersrepresent DNA units, bold upper case letters represent beta-D-oxy-LNAunits, all LNA C are 5′methyl C and all internucleoside linkages arephosphorothioate linkage.

7. The method according to any one of claims 1 to 6, wherein step d)further comprises the measurement of extracellular kidney injurymolecule-1 (KIM-1) protein or intracellular mRNA levels, wherein anincrease in KIM-1 levels are indicative of a nucleic acid molecule whichis, or is predicted to be, associated with nephrotoxicity.

8. The method according to embodiment 7, wherein a level of KIM-1protein above 200% relative to the saline or non-toxic reference valueis predicative of nephrotoxicity of the nucleic acid molecule.

9. The method according to embodiment 7, wherein a level of KIM-1 mRNAabove 1000% relative to the saline or non-toxic reference value ispredicative of nephrotoxicity of the nucleic acid molecule

10. The method according to embodiment 7 to 9, wherein the increase inKIM-1 is predicative of nephrotoxicity for a nucleic acid molecule evenif the EGFR mRNA level is not decreased.

11. The method according to any one of embodiments 1 to 10 wherein theculture media in step a) comprises at least 4 ng/ml of epidermal growthfactor (EGF) and step d) further comprises the measurement ofintracellular adenosine triphosphate (ATP) levels; wherein a decrease inintracellular ATP levels is indicative of a drug substance which is, oris predicted to be, associated with nephrotoxicity.

12. The method according to embodiment 11, wherein a level ofintracellular ATP below 80% relative to the saline or non-toxicreference value is predicative of nephrotoxicity of the drug substance.

13. The method according to any one of embodiment 1 to 12, wherein thecells expressing EGFR is selected from the group consisting ofepithelial cell, endothelial cell and mesenchymal cells, neuroectodermalcells.

14. The method according to any one of embodiment 1 to 13, wherein thecells expressing EGFR is an epithelial cell culture.

15. The method according to embodiment 13 or 14, wherein the cells arein the form of a primary cell culture selected from the group consistingof rodent primary cells, such as mouse or rat primary cells; pig (e.g.minipig) cells and primate primary cells, such as monkey (e.g.cynomolgus monkey), human primary cells.

16. The method according to embodiment 15, wherein the primary cellculture is obtained from a rat or a human epithelial cells.

17. The method according to any one of embodiments 14 to 16, wherein theepithelial cell culture is a kidney epithelial cell culture,gastrointestinal tract epithelial cell culture or a lung epithelial cellculture.

18. The method according to embodiment 17, wherein the kidney epithelialcell is selected from the group consisting of proximal tubule epithelialcells, distal tubule epithelial cells and collecting duct epithelialcells.

19. The method according to any one of claim 17 or 18, wherein the cellculture is made from human PTEC or rat PTEC cells.

20. The method according to any one of claims 1 to 14 or 17 or 18,wherein the cells expressing EGFR are cultured from an immortalized cellline.

21. The method according to embodiment 20, wherein cell culture isobtained from the group consisting of human PTEC-TERT-1, ciPTEC, CACO2,HK-2, NKi-2 or human A549 cell lines.

22. The method according to any one of claims 1 to 21, wherein the cellculture media contains at between 5 and 15 ng/ml, such as between 8 and15 ng/ml of epidermal growth factor (EGF), such as around 10 ng/ml.

23. The method according to any one of claims 1 to 21, wherein theperiod of incubation with the nucleic acid molecule is between 2 and 6days, such as around 3 days.

24. The method according to any one of claims 1 to 23, wherein thenucleic acid molecule is selected from a RNAi agent, an antisenseoligonucleotide or an aptamer.

25. The method according to any one of claims 1 to 24, wherein thenucleic acid molecule is administered to the cell culture in thepresence of a transfection agent.

26. The method according to any one of claims 1 to 23, wherein thenucleic acid molecule, in particular a single stranded oligonucleotide,is administered to the cell culture in the absence of a transfectionagent, i.e. by the process referred to as gymnosis.

27. The method according to any one of claims 1 to 26, wherein thenucleic acid molecule comprises one or more 2′ sugar modifiednucleosides.

28. The method according to embodiment 27, wherein the one or more 2′sugar modified nucleoside is independently selected from the groupconsisting of 2′-O-alkyl-RNA, 2′-O-methyl-RNA, 2′-alkoxy-RNA,2′-O-methoxyethyl-RNA, 2′-amino-DNA, 2′-fluoro-DNA, arabino nucleic acid(ANA), 2′-fluoro-ANA and LNA nucleosides.

29. The method according to embodiment 27 or 28, wherein the one or more2′ sugar modified nucleoside is a LNA nucleoside.

30. The method according to embodiment 28 or 29, wherein the LNAnucleoside is selected from beta-D-oxy-LNA, alpha-L-oxy-LNA,beta-D-amino-LNA, alpha-L-amino-LNA, beta-D-thio-LNA, alpha-L-thio-LNA,(S)cET, (R)cET, beta-D-ENA or alpha-L-ENA

31. The method according to any one of claims 1 to 30, wherein thenucleic acid molecule comprises at least one modified internucleosidelinkage.

32. The method according to embodiment 31, wherein the internucleosidelinkages within the contiguous nucleotide sequence are phosphorothioateinternucleoside linkages.

33. The method according to any one of claims 1 to 32, wherein thenucleic acid molecule is an antisense oligonucleotide capable ofrecruiting RNase H.

34. The method according to embodiment 33, wherein the antisenseoligonucleotide is a gapmer.

35. The method according embodiment 33 or 34, wherein the antisenseoligonucleotide is a gapmer of formula 5′-F-G-F′-3′, where region F andF′ independently comprise 1-7 modified nucleosides and G is a regionbetween 6 and 16 nucleosides which are capable of recruiting RNaseH.

36. The method according to any one of claims 1 to 35, wherein the invivo nephrotoxicity is acute kidney injury and/or tubular degradation.

37. A method for selecting one or more nucleic acid molecules for invivo administration to a mammal, from a library of nucleic acidmolecules, said method comprising the steps of

-   -   a. obtaining a library of nucleic acid molecules;    -   b. administering each member of the library of nucleic acid        molecules to a cell culture expressing epidermal growth factor        receptor (EGFR), such as per any one of claims 13 to 22;    -   c. culturing the cells in vitro for a period of time, such as        per embodiment;    -   d. measuring the amount of intracellular EGFR mRNA for each        nucleic acid molecule, such as per any one of the claims 1 to        12; and    -   e. selecting one or more nucleic acid molecules wherein %        reduction in EGFR relative to a reference value is above 80%.

38. The method according to embodiment 37, wherein the therapeutic indexof the selected nucleic acid molecule is decreased when compared to atoxic reference substance or parent nucleic acid molecule.

39. The method according to embodiment 37 or 38, wherein the library ofnucleic acid molecules is a library of nucleic acid molecules as per anyone of claims 25 to 35.

40. The method according to any one of claims 37 to 39, wherein thelibrary of nucleic acid molecules is a library of antisenseoligonucleotides.

41. The method according to any one of claims 37 to 40, wherein thelibrary of nucleic acid molecules is a library of nucleic acid moleculevariants (child nucleic acid molecules) of a parent nucleic acidmolecule, wherein the parent nucleic acid molecule is toxic, such asnephrotoxic, and wherein step d) identifies one or more nucleic acidmolecule variants which are less toxic than the parent nucleic acidmolecule; wherein the nucleic acid molecule variants retain thenucleobase sequence of the parent nucleic acid molecule.

42. The method according to any one of claims 37 to 41, wherein thelibrary of nucleic acid molecule variants comprises a population ofchild nucleic acid molecules which differ by virtue of the design fromthe parent nucleic acid molecule.

43. The method according to embodiment 37 to 42, wherein the nucleicacid molecule variants are antisense oligonucleotides and differ fromthe parent antisense oligonucleotide in one or more design parametersselected from the group consisting of

-   -   i. presence of one or more stereodefined phosphorothioate        internucleoside linkages;    -   ii. change in gap size;    -   iii. introduction of a gap breaker;    -   iv. change of wing size;    -   v. change in 2′ sugar modified nucleosides in the wings; and    -   vi. mixed wing gapmers with at least two different 2′ modified        nucleosides in the wings, such as LNA nucleosides and 2′        substituted nucleosides, in particular MOE.

44. The method according to embodiment 37 to 43, wherein the selectionone or more drug substances for in vivo administration to a mammal isfurther based on results from an in vitro immunotoxicity assay and/or anin vitro hepatotoxicity assay which are suitable for predicting in vivoimmunotoxicity and/or in vivo hepatotoxicity.

45. A nucleic acid molecule obtained by the method according to any oneof claims 37 to 43.

46. A pharmaceutical composition comprising the nucleic acid molecule ofembodiment 44 and a pharmaceutically acceptable diluent, solvent,carrier, salt and/or adjuvant.

47. The nucleic acid molecule of embodiment 44 or the pharmaceuticalcomposition of embodiment 46 for use in a medicine.

EXAMPLES

Methods and Materials

Compounds

SEQ In vivo Comp ID nephro- ref Target Compound 5′ → 3′ NO toxicity  1-1Scramble CGTcagtatgcgAATc  1 innocuous  2-1 PCSK9 AATgctacaaaaCCCA  2low  3-1 PCSK9 TGCtacaaaacCCA  3 medium  4-1 PCSK9 GCtgtgtgagcttGG  4high  5-1 Myd88 TAAggcaatcaagGTA  5 medium  6-1 Myd88 CAAaggaaacacaCAT 6 innocuous  7-1 Myd88 CAAatgctgaaacTAT  7 innocuous  8-1 Myd88ACTgctttccactCTG  8 high  8-2 Myd88 ACTgc_(s)tttc_(s)cac_(s)tCTG  8 n.d 8-3 Myd88 ACTgc_(r)tttc_(r)cac_(r)tCTG  8 n.d  9-1 Myd88GCCtcccagttccTTT  9 low/medium 10-1 Myd88 CACattccttgctCTG 10 medium10-2 Myd88 CACatt_(s)cctt_(s)gct_(s)CT_(s)G 10 n.d 10-3 Myd88CACatt_(r)cctt_(r)gct_(r)CT_(r)G 10 n.d 10-4 Myd88CACatt_(s)c_(s)ctt_(s)g_(s)ctCTG 10 n.d 10-5 Myd88CACatt_(r)c_(r)ctt_(r)g_(r)ctCTG 10 n.d 11-1 Myd88 TGCtcaacatcAAG 11medium 12-1 Myd88 TTAcacttgacCCA 12 high 13-1 Myd88 TTTacacttgaCCC 13medium 14-1 Myd88 GTCagaaacaaccACC 14 high 15-1 BCL11A CTAtgtgttccTGT 15medium/high 16-1 BCL11A CGTttgtgctcgaTAA 16 medium/high 17-1 BCL11ACGTttgtgctcgATA 17 high 18-1 BCL11A ATTgcattgtttcCGT 18 low 19-1 BCL11ACATtgcattgtttCCG 19 low/medium 19-2 BCL11A CATtgcattgttTCCG 19 high 20-1SGLT2

catgagct

20 low/medium n.d. = not determined

For the compounds lower case letters represent DNA units, upper caseletters represent beta-D-oxy-LNA units. All LNA C units are 5′ methyl Cand all internucleoside linkages are phosphorothioate linkage. Rpstereodefined phosphorothioate linkage are indicated by subscript r. Spstereodefined phosphorothioate linkage are indicated by subscript s.Bold, italic lower case letters represent MOE units. In compound 20-1all C units (DNA and MOE) are 5′ methyl C. The SEQ ID NO refers to thenucleobase sequence of the compound.

Measuring In Vivo Nephrotoxicity

Purpose bred Wistar Han Crl:WI(Han) male rats obtained from CharlesRiver Laboratories at 7 to 8 weeks of age were divided into groups of 4(table 1, exp. A) or 8 (table 1, exp. B) based on body weight andacclimatized for at least 5 days before dosing. The animals were housedunder standard environmental conditions (22±2° C., relative humidity50±20%, a light/dark cycle 12 h/12 h, pelleted food and water adlibitum), were offered enriched environment in an AAALAC accreditedfacility and were regularly and carefully monitored. All procedures werein accordance with the respective local regulations and according to theanimal permissions granted by the Institutional Animal Care and UseCommittee. Test compounds were formulated in isotonic sterile saline,sterile filtered (0.22 μm), and dosed at 40 mg/kg on days 1 and 8 (2.5mL/kg) in the intrascapular region. Control group animals receivedsaline as vehicle control. On day 15 animals were orally administeredtap water (10 mL/kg), and urine was collected on ice for 6 hours inmetabolic cages. Urine protein levels (Aution Max AX-4280) and urinaryrenal injury biomarkers were measured (Multiplex MAP Rat Kidney ToxicityMagnetic Bead Panel 2). On day 15 the rats were sacrificed by anintraperitoneal injection of pentobarbital and exsanguinated. Kidneycortex samples were collected and fixed by immersion in 10% neutralbuffered formalin, embedded in paraffin, sectioned to 5 μm and stainedwith hematoxylin and eosin (H&). The H&E sections were then scanned at20× magnification using an Aperio ScanScope AT (Leica Biosystems)scanner and pictures were then captured via ImageScope software from thescanned images. One or more of the following parameters were assessedkidney weight, serum creatinine, urine protein, KIM-1 protein in urine,KIM-1 mRNA, kidney degeneration/regeneration and relative tubulotoxicitygrade.

Cell Cultures

Human Primary Proximal Tubule Epithelial Cells (PTEC) Cell Culture

Primary PTEC (Science Cell Research Labotories catalog #4100) werecultured according to the manufacturer's instructions in PTEC medium[DMEM/F12 without phenol red (ThermoFisher Scientific 11039021)containing 1% Penicillin-Streptomycin solution (ThermoFisher Scientific15140122), 10 mM HEPES (ThermoFisher Scientific 15630056), 5 ug/mlinsulin and 5 ug/ml transferrin and 8.65 ng/ml sodium selenite (all froma 100× concentrated stock solution, ThermoFisher Scientific 41400045),100 nM hydrocortisone (Sigma H6909), 3.5 ug/ml ascorbic acid (SigmaA4403), 25 ng/ml prostaglandin E1 (Sigma P5516), 3.25 pg/mltriiodo-L-thyronine (Sigma T6397), 10 ng/ml human recombinant epidermalgrowth factor (EGF, R&D Systems 236-EG-200), and 100 μg/ml Geneticin(G418 sulfate, ThermoFisher Scientific 10131027)].

Prior to treatment with oligonucleotides the primary PTEC cells wereseeded into collagen I-coated 96-well plates (Corning, 356407) at adensity of 40 000 and 20 000 cells/well respectively in PTEC medium andgrown until confluence.

Human PTEC-TERT1 Cell Culture

PTEC-TERT1 (Evercyte GmbH, Austria) were cultured according to themanufacturer's instructions in PTEC medium [DMEM/F12 without phenol red(ThermoFisher Scientific 11039021) containing 1% Penicillin-Streptomycinsolution (ThermoFisher Scientific 15140122), 10 mM HEPES (ThermoFisherScientific 15630056), 5 ug/ml insulin and 5 ug/ml transferrin and 8.65ng/ml sodium selenite (all from a 100× concentrated stock solution,ThermoFisher Scientific 41400045), 100 nM hydrocortisone (Sigma H6909),3.5 ug/ml ascorbic acid (Sigma A4403), 25 ng/ml prostaglandin E1 (SigmaP5516), 3.25 μg/ml triiodo-L-thyronine (Sigma T6397), 10 ng/ml humanrecombinant epidermal growth factor (EGF, R&D Systems 236-EG-200), and100 μg/ml Geneticin (G418 sulfate, ThermoFisher Scientific 10131027)].

Prior to treatment with oligonucleotides the PTEC-TERT1 were seeded intocollagen I-coated 96-well plates (Corning, 356407) at a density of 40000 and 20 000 cells/well respectively in PTEC medium and grown untilconfluence.

Rat Primary Proximal Tubule Epithelial Cells (PTEC) Cell Culture

Proximal renal tubule epithelial cells were prepared by a simplifiedprocedure of that described by Bruce et al, Methods Mol Biol 1001, 53-64(2013). Kidneys were removed from one anesthetized male Wistar rat (age:10-18 weeks) and were washed in cold 1× concentrated Hanks balanced saltsolution (HBSS, ThermoFisher Scientific 14065049). The organs weredecapsulated, and the outer stripes of the kidney cortex without medullawere cut into 1 to 2 mm³ pieces. Tissue pieces were digested in 25 ml ofHBSS containing 2 mg/ml collagenase A (Roche 10103586001) and 10 ug/mlDNAse I (Roche 10104159001) at 37° C. for 60 min. with continuous gentleshaking. The reaction was stopped by addition of 25 ml of ice-cold HBSScontaining 2% bovine serum albumin (BSA, Fraction V, Roche 10735086001).Dissociated tissue was filtered consecutively first through a 200 μmcell strainer (pluriSelect 43-50200) and subsequently through a 100 umcell strainer (pluriSelect 43-50100); the filtrate containing singlecells and tubule fragments was washed and centrifuged three times (100g) in HBSS solution without MgCl2 and CaCl2) (HBSS−/−, ThermoFisherScientific 14175053). The sediment was resuspended in 25 ml HBSS−/− andmixed with 25 ml of 30% OptiPrep solution [mixture of 5 volumes of 60%OptiPrep density gradient media (Sigma D1556), 4 volumes of H2O sterile,and 1 volume of 10× concentrated phosphate buffered saline without MgCl2and CaCl2) (PBS−/− ThermoFisher Scientific 14200-067) to obtain asuspension of cells and tubular fragments in 15% OptiPrep. Thesuspension was centrifuged (800 g, at room temperature for 20 min). Thecell band in the upper third of the tube was harvested; cells in thesediment at the bottom of the tube were discarded. Harvested cells werewashed and centrifuged three times (400 g, at room temperature for 5min.) in rat PTEC medium [1 volume DMEM (ThermoFisher Scientific11966025), 1 volume Ham's F-12 nutrient mix (ThermoFisher Scientific21765029), 2% fetal bovine serum (FBS, ThermoFisher Scientific16000044), 1% Penicillin-Streptomycin solution (ThermoFisher Scientific15140122), 10 mM HEPES (ThermoFisher Scientific 15630056), 5 ug/mlinsulin and 5 ug/ml transferrin and 8.65 ng/ml sodium selenite (all froma 100× concentrated stock solution, ThermoFisher Scientific 41400045),100 nM hydrocortisone (Sigma H6909), 3.5 ug/ml ascorbic acid (SigmaA4403), 25 ng/ml prostaglandin E1 (Sigma P5516), 3.25 μg/mltriiodo-L-thyronine (Sigma T6397), and 10 ng/ml rat recombinantepidermal growth factor (EGF, R&D Systems 3214-EG-100/CF)]. After thefinal centrifugation step, the cell preparation was resuspended in 50 mlof rat PTEC medium and distributed into three collagen I-coated 150 cm²cell culture flasks (Corning 354486) followed by incubation for 4 dayswith a change of medium after the first 2 days.

Prior to treatment with oligonucleotides the expanded rat PTEC cellswere harvested from the culture flasks by detachment with trypsin-EDTAsolution (ThermoFisher Scientific 25200056), washed in rat PTEC mediumand seeded into collagen I-coated 96 well plates (Corning, 356407) at adensity of 36 000 cells/well in rat PTEC medium for growth untilconfluence.

CACO2 (Human Epithelial Colorectal Adenocarcinoma Cells)

CACO2 (ATCC, HTB-37) were cultivated according to the manufacturer'sinstructions in CACO2 medium [DMEM/F12-Glutamax, non-essential aminoacid (1%), penicillin/streptomycin (1%) (Gibco, 31331-028)(Gibco,11140-035)(Gibco: 15140-122), 20% FBS (Gibco, 16000-044] for 2 passagesthen switched to CACO2 medium containing 10% FBS.

Prior to treatment with oligonucleotides the CACO2 cells were seededinto 96-well plates at 10 000 cells per well and grown until confluence.

A549 Cells (Adenocarcinomic Human Alveolar Basal Epithelial Cells)

A549 cells (ATCC CCL-185) were cultivated according to themanufacturer's instructions in A549 medium [F-12K Nut Mix (Gibco,21127-022), 1% penicillin/streptomycin (Gibco: 15140-122), 10% FBS(Gibco 16000-044)].

Prior to treatment with oligonucleotides the A549 cells were seeded in96 well plates at 3000 cells per well in A549 regular medium and grownuntil 60% confluence.

For lipofection, cells were transfected with 1 or 10 ng ofoligonucleotide using lipofectamin (Invitrogen, 11668-019) according tothe manufacturer's instructions. After 24 hours the medium was changedfor A549 regular medium containing 10 ng/ml EGF and 2% FCS. After 72hours the cells were harvested and EGFR mRNA was measured and thesupernatant was stored at −20° C. for KIM-1 analysis.

For gymnosis the cells were grown to confluence and subjected to thegymnosis protocol below.

Oligonucleotide Treatment Using Gymnosis

Oligonucleotides were dissolved in PBS and added to the cell culture atthe given concentration, such as 10, 30 or 100 μM. The total volume ofeach well was 100 μl. PBS served as vehicle control.

When intracellular ATP was to be analyzed following the oligonucleotidetreatment the medium was changed every 3 days including addition of thesame concentration of oligonucleotide as day 0.

When extra cellular markers, like KIM-1, were analyzed following theoligonucleotide treatment the medium was collected and stored at −20° C.until further analysis. Cells that were grown beyond 3 days of the firstoligonucleotide treatment had the medium collected and replaced every 3days incl. fresh oligonucleotide. The collected medium was stored at−20° C.

ATP Assay

For the determination of intracellular ATP levels the CellTiter-Glo®Luminescent Cell Viability Assay (G7571, Promega Corporation, MadisonWI, USA) was used according to the manufacturer's instructions. Eachsample was tested in triplicate. Unless indicated otherwise theintracellular ATP levels were measured at day 9 after oligonucleotidetreatment. A decrease in ATP levels below 80% in relation to vehicle wasconsidered as predictive for nephrotoxicity.

EGFR and PCSK9 mRNA Assay

Cells were collected by replacing culture medium with 100 μl/well of1×RNA lysis mixture (QuantiGene® Sample Processing Kit, QS0101). RNAlysis mixtures were kept at −80° C. until analysis. 20 μl of lysateswere mixed with an mRNA-capture magnetic beads sets (for human cellsPanomics QuantiGene® Plex Set #12871 and for rat cells PanomicsQuantiGene® Plex Set #31357), incubated overnight, processed forbranched DNA amplification and analyzed according to the manufacturer'sinstructions (Panomics QuantiGene® Plex Assay kit, QP1015). The PPIBprobe was used as housekeeping gene for normalization. AverageFluoresence Intensity (FI) and standard deviation of 3 biologicalreplicates were calculated and normalized on vehicle control. A decreasein EGFR mRNA levels below 80%, preferably below 75%, in relation tovehicle was considered as predictive for nephrotoxicity.

KIM-1 Assay

For analysis of human and rat KIM-1, cell supernatants were thawed onice, diluted 1:2 and 1:10 in sample dilution buffer (BioRad catalog#M60-009RDPD) and analyzed by Luminex-based ELISA using human KIM-1beads (Human Kidney Injury panel 4, Millipore HKI4MAG-99K-KIM1) or ratKim-1 beads (Milliplex Rat Kidney toxicity RKTX1MAG-37K-Kim1) followedby analysis using the Bio-Plex® 200 Systems (BioRad) according to themanufacturer's instructions. Data are reported as mean concentrationsand standard deviations of triplicate wells. An increase of KIM-1protein levels above 200% in relation to vehicle was considered aspredictive for nephrotoxicity.

KIM-1 mRNA levels were measured according to the same procedure as theEGFR mRNA levels. In rats Panomics QuantiGene® Plex Set #31357 was used,where HAVCR1 corresponds to KIM-1. An increase of KIM-1 mRNA levelsabove 1000% in relation to vehicle was considered as predictive fornephrotoxicity

Example 1 Morphological Changes of Cell Cultures Upon OligonucleotideTreatment

The present example evaluates the effect on the morphology of a cellculture following oligonucleotide treatment.

PTEC-TERT1 cells were cultures as described in the “Materials andmethods” section. To mimic the physiological exposure of renal tubulesto circulating naked oligonucleotide (i.e. without assistance ofdelivery technology, herein termed gymnosis or gymnotic delivery),confluent monolayers of PTEC-TERT1 cells were exposed to an aqueoussolution of 100 μM oligonucleotide. The medium was replaced every threedays including 100 μM fresh oligonucleotide in the fresh medium.

After 7 days of treatment the morphological alterations in the treatedcell cultures were investigated under bright field microscopy. FIG. 1shows that PTEC-TERT1 cells treated with oligonucleotide compound 1-1(innocuous in vivo) did not show any morphological changes compared tosaline treated cells. Cells treated with oligonucleotide compound 3-1(medium in vivo tox) formed irregular domes and vacuoles whereasPTEC-TERT1 cells treated with oligonucleotide compound 4-1 (high in vivotox) adopted a flattened and stable appearance. Both the toxic compoundsshow morphological changes, however the less toxic compound 3-1 producemore significant morphological changes than the highly toxic compound4-1.

In conclusion toxic oligonucleotides affect the morphology, the severityof the toxicity can however not be predicted using morphologicalchanges.

Example 2 PCSK9 Targeting Oligonucleotides and their Effects on EGFRmRNA Expression in PTEC-TERT1 Cells

The present example evaluates the effect on the biomarker, EGFR, incells exposed to oligonucleotides in a time course from 8 hours to 6days. The effect on the target nucleic acid, PCSK9 was also analyzed toshow efficacy of the compounds.

The immortalized PTEC-TERT-1 cell line was cultured according to theconditions described in the “Materials and methods” section. Atconfluence the cells were treated with oligonucleotide as described inthe “Materials and methods” section, in concentrations of 100 μM.

The EGFR mRNA level and PCSK9 mRNA level in the cells was measuredaccording to the assay as described in the “Materials and methods”section.

The results are shown in tables 2 and 3 below and represent the averageof three identical treatments.

TABLE 2 EGFR mRNA concentrations as % of saline after oligonucleotidetreatment for the indicated time 8 h 24 h 48 h 72 h Day 6 In vivo toxComp # EGFR SD EGFR SD EGFR SD EGFR SD EGFR SD saline 100 12 100 29 1004 100 10 100 11 Innocuous 1-1 98 5 99 6 99 6 97 8 106 5 low 2-1 98 9 1005 116 3 122 12 129 8 Medium 3-1 90 7 94 6 89 5 89 11 74 5 high 4-1 96 1376 3 73 4 56 2 46 5

TABLE 3 PCSK9 mRNA concentrations as % of saline after oligonucleotidetreatment for the indicated time 8 h 24 h 48 h 72 h Day 6 In vivo toxComp # PCSK9 SD PCSK9 SD PCSK9 SD PCSK9 SD PCSK9 SD saline 100 15 1003.3 100 5 100 12 100 4 Innocuous 1-1 93 1.4 102 1.4 105 2.4 126 14.6 9011.2 low 2-1 29 0.2 3.5 1.2 1.8 0.3 1.1 0.2 0.7 0.3 Medium 3-1 40 1.66.7 1.6 2.5 0.3 0.5 0.2 0.5 0.2 high 4-1 66 4.0 32 1.2 10 0.8 3.5 1.31.9 1.1

From this it can be seen that already after 48 hours the highly toxicoligonucleotide (comp 4-1) starts to down regulate EGFR, whereas themedium toxic compound (comp 3-1) shows effects on EGFR down regulationafter 6 days. The compounds targeting PCSK9 (comp 2-1, 3-1 and 4-1) allshow effective knock down of PCSK9 after 24 hours. The low toxiccompound 2-1 actually appears to be the most effective in downregulating PCSK9, so the down regulation of PCSK9 does not appear tocontribute to the toxicity of the oligonucleotides.

A further investigation of the compounds that down regulate EFGR (comp3-1 and 4-1) showed that these oligonucleotides contained only onemismatch to the EGFR premRNA, whereas the innocuous compounds did notshow any significant match (complementarity) to the EGFR mRNA. The downregulation of the EGFR mRNA could therefore theoretically be due tooff-target effects, we do however not believe this is the case based onthe observations found in Example 3 below.

Example 3 PCSK9, MYD88 and Bcl11A Targeting Oligonucleotide Effects onExtracellular

The present example evaluates whether the findings in example 2 can bereproduced for a larger set of oligonucleotides directed to othertargets. In the same study the biomarker KIM-1, EGF and ATP was measuredas well.

The immortalized PTEC-TERT-1 cell line was cultured according to theconditions described in the “Materials and methods” section. Atconfluence the cells were treated with oligonucleotide as described inthe “Materials and methods” section, in concentrations of 10, 30 or 100μM.

For EGFR and KIM-1 measurements were performed at day 6. EGFR mRNA andKIM-1 protein levels were measured according to the assays described inthe “Materials and methods” section.

The results are shown in tables 4 below, and represent the average ofthree identical treatments.

TABLE 4 EGFR mRNA level in the cells and KIM-1 levels in culture mediaat day 6 after oligonucleotide treatment and intracellular ATP levelswas measured at day 9 after oligonucleotide treatment. In vivo Day 6 Day6 Day 9 grade Comp Conc EGFR KIM-1 ATP of toxicity # μM % saline SD %saline SD % saline SD High 4-1 30 60 6.5 43 6.4 n.d 100 44 2.0 1 2.6n.d. Innocuous 6-1 10 105 1.6 140 13 93 4 30 102 3.6 150 6 99 3 100 1153.0 122 8 101 3 Innocuous 7-1 10 98 9.9 159 4 113 7 30 98 17.0 127 3 1146 100 97 9.7 120 6 107 1 High 8-1 10 44 4.2 78 29 59 3 30 27 0.3 63 1 477 100 21 1.0 54 1 43 1 Medium 9-1 10 64 0.6 151 7 99 2 30 53 8.8 129 8101 2 100 50 0.7 116 29 103 4 Medium 10-1  10 80 5.9 123 17 87 2 30 686.2 77 1 66 6 100 66 7.2 65 4 60 5 Medium 11-1  10 37 0.6 149 13 136 230 29 11.0 111 10 135 6 100 18 0.6 82 8 119 4 High 12-1  10 102 3.8 32537 132 2 30 104 7.9 379 20 133 1 100 94 5.2 289 26 90 4 Medium 13-1  10115 6.2 268 46 124 10 30 115 7.6 242 51 122 5 100 114 6.6 252 17 114 2Medium 15-1  10 68 2.5 43 3 101 2 30 59 5.2 34 3 92 0 100 65 3.3 25 2 522 Mild tox 18-1  10 96 1.8 188 17 121 1 30 104 10.2 193 21 112 4 100 9810.8 181 13 105 0 Mild tox 19-1  10 86 7.6 120 11 100 2 30 76 1.4 129 2490 2 100 64 2.3 77 15 76 1 Medium 16-1  10 91 1.4 160 10 123 1 30 75 5.2164 15 122 1 100 72 4.4 129 16 113 0 High 19-2  10 69 3.4 75 11 67 1 3056 5.2 57 16 54 2 100 66 8.1 28 4 40 1 High 17-1  10 77 9.1 167 20 124 130 64 7.9 202 4 116 2 100 64 22.2 140 23 96 1

From table 4 it can be seen that the EGFR biomarker is capable ofpredicting 10 out of 13 toxic compounds at the 100 μM oligonucleotidedose. Of these oligonucleotides compound 13-1 and 15-1 have twomismatches to human EGFR premRNA. Compound 13-1 does not affect the EGFRmRNA levels so two mismatches in this compound most likely results in abinding affinity that is too low to result in cleavage of the EGFRpremRNA. Compounds 8-1, 9-1, 10-1, 11-1, and 16-1 all reduce the levelof EGFR mRNA, none of these compounds are complementary to the humanEGFR premRNA to a level where they would be expected to cleave thetarget (they have more than 3 mismatches to human EGFR premRNA).Consequently, the down regulation of EGFR mRNA by these compounds cannotbe attributed to off-target effects and therefore establishes that EGFRmRNA is a relevant biomarker for the prediction of nephrotoxicity ofoligonucleotides independent of their level of complementarity to EGFRpremRNA.

KIM-1 only predicts 2 out of the 13 toxic compounds. However, these arethe compounds that were not caught be the EGFR biomarker, indicatingthat KIM-1 can supplement the predictions made with EGFR as biomarker.

ATP as biomarker is capable of predicting 5 of the 12 toxic compounds.Consequently, the ATP biomarker can be used to supplement the EGFRbiomarker.

The results are also summarized in FIG. 2 .

Example 4 PCSK9 and Bcl11A Targeting Oligonucleotides and their Effectson EGFR and KIM-1 Expression in Rat Primary PTEC Cells

The present example evaluates whether the findings in example 2 and 3can be reproduced in a cell line from a different species.

A cell culture of primary rat PTEC cells were made and culturedaccording to the conditions described in the “Materials and methods”section. At confluence the cells were treated with oligonucleotide asdescribed in the “Materials and methods” section, in concentrations of1, 10, or 100 μM.

For EGFR and KIM-1 measurements were performed at day 3. EGFR mRNA andKIM-1 protein levels were measured according to the assays described inthe “Materials and methods” section.

The results are shown in tables 5 below, and represent the average ofthree identical treatments.

TABLE 5 EGFR and KIM-1 levels at day 3 after oligonucleotide treatment.In vivo grade of Conc EGFR KIM-1 mRNA KIM-1 prot toxicity Comp # μM %1-1 SD % 1-1 SD % 1-1 SD Innocuous  1-1 1 100 7.2 100 18.7 100 13.0 10100 11.5 100 32.6 100 9.0 100 100 9.4 100 20.6 100 10.4 medium  3-1 1 679.0 1321 106.7 262 48.3 10 84 5.9 3622 93.4 433 101.8 100 99 17.5 2874486.7 65 7.5 High  4-1 1 23 4.4 213 71.4 99 20.8 10 12 1.0 254 98.3 9810.5 100 11 7.3 67 31.7 92 Medium/high 15-1 1 39 7.6 299 144.0 103 27.410 29 7.0 354 81.2 114 23.4 100 68 5.1 151 110.7 152 50.1 Medium/high16-1 1 83 18.6 188 75.1 122 33.5 10 90 18.0 356 123.9 140 42.1 100 10418.0 226 69.5 141 43.6 High 17-1 1 83 13.1 306 80.0 108 21.2 10 83 18.4405 116.7 151 32.7 100 95 16.5 422 115.8 184 55.0 Low 18-1 1 64 5.8 42369.6 137 22.2 10 42 11.4 556 105.9 159 22.4 100 51 12.2 526 474.7 17451.1 Low/medium 19-1 1 53 10.5 1676 480.0 185 46.4 10 48 17.4 1991 781.4231 72.1 100 35 14.7 1437 378.5 345 52.2 High 19-2 1 25 10.5 5139 1462.9502 81.3 10 10 7.7 5361 1106.2 712 88.8 100 6 10.3 2367 606.9 566 149.7

Primary rat PTEC cells are quite sensitive to oligonucleotide treatmentin general and it appears that doses of 100 μM oligonucleotide affectthe cells to an extent where the assay becomes less predictable. Toaccount for the sensitivity of the rat PTEC cells the data in table 5have been normalized to the non-toxic toxic reference antisenseoligonucleotide indicated as comp 1-1. Using a reduction in the EGFRlevel as a biomarker the assay is capable of predicting 6 out of 8 toxiccompounds at the 1 μM dose (comp 3-1, 4-1 15-1, 18-1, 19-1 and 19-2).None of the compounds have below 2 mismatches to rat EGFR premRNAconsequently the effects are not expected to be off-target effects.

Increases in KIM-1 levels are significantly different at the mRNA leveland the protein level. For KIM-1 mRNA levels the threshold for anindication of toxicity was set at a 10 fold increase over the innocuouscompound 1-1. The threshold for an indication of toxicity when measuringKIM-1 protein level was set at a 2 fold increase over the innocuouscompound 1-1. With these thresholds 3 out of the 8 toxic compounds werepredicted as toxic (comp 3-1, 19-1 and 19-2) indicating that KIM-1 cansupplement the predictions made with EGFR as biomarker for somecompounds.

From this it can be seen that the EGFR biomarker in rat primary PTECcells can be used to predict the in vivo toxicity of oligonucleotides.The cells are however more sensitive to oligonucleotide treatment, andit is therefore recommended to perform the evaluation in relation to anon-toxic toxic reference antisense oligonucleotide and at loweroligonucleotide concentrations than for human PTEC and PTEC-TERT1 cells.

Example 5 Oligonucleotide Effects on EGFR in CACO2 Cells

In the following example it was investigated whether a cell line thatdoes not originate from the kidney and which does not have an active EGFconsumption but which does express EGFR can be used to predictnephrotoxicity of an oligonucleotide.

The immortal CACO2 cell line was cultivated as described in the“Materials and methods” section without the presence of EGF to themedium. At confluence the cells were treated with oligonucleotide usinggymnosis as described in the “Materials and methods” section, inconcentrations of 1, 10 or 100 μM.

The cells were harvested at day 6, and the EGFR and PCSK9 mRNA level wasmeasured according to the assay described in the “Materials and methods”section.

The results are shown in table 6 below, and represent the average ofthree identical treatments.

TABLE 6 EGFR and PCSK9 mRNA levels in CACO2 cells at day 6 afteroligonucleotide treatment. ATP levels after 9 days treatment. In vivoEGFR PCSK9 ATP grade Comp Conc % % % of toxicity # μM saline SD salineSD saline SD saline 0 100 4.4 100 5.2 100 5.2 Innocuous 1-1 1 74 5.9 995.5 108 9.0 10 71 7.0 108 9.6 98 13.3 100 83 5.4 145 5.5 93 15.4 low 2-11 80 7.6 39 3.2 104 9.6 10 68 1.4 10 1.3 102 14.4 100 57 4.8 3 0.1 10310.2 Medium 3-1 1 91 2.5 37 3.0 107 10.5 10 72 5.7 9 1.3 97 4.5 100 451.4 2 0.1 97 9.0 High 4-1 1 86 11.4 75 16.7 102 6.8 10 36 4.6 15 2.0 1006.7 100 15 4.9 4 0.8 82 7.3

From this it can be seen that at 10 and 100 μM oligonucleotide thenephrotoxic compounds (comp 3-1 and 4-1) can be identified using CACO2cells. The conditions of this example does however not allow generatepredictive results for ATP as a biomarker.

Example 6 Oligonucleotide Effects on EGFR in A549 Cells

In this example it was tested whether another cell line that does notoriginate from the kidney which do have an active EGF consumption andexpress EGFR can be used to predict nephrotoxicity of anoligonucleotide.

A549 were cultivated as described in the “Materials and methods”section. At confluence the cells were treated with oligonucleotide usinggymnosis as described in the “Materials and methods” section, inconcentrations 100 μM.

The cells were harvested at day 2 and the EGFR and PCSK9 mRNA level wasmeasured according to the assay described in the “Materials and methods”section.

The results are shown in table 7 below, and represent the average ofthree identical treatments.

TABLE 7 EGFR and PCSK9 mRNA levels in A549 cells at day 2 after gymnoticoligonucleotide treatment. In vivo grade of Conc EGFR PCSK9 toxicityComp # μM % saline SD % saline SD saline 0 100 5.7 100 0.2 Innocuous 1-1100 97 2.5 93 3.6 Medium 3-1 100 83 1.2 0 0.9 High 4-1 100 33 5.0 1 0.2High 8-1 100 32 5.4 145 9.7

From this it can be seen that at 100 μM oligonucleotide the highlynephrotoxic compounds (comp 4-1 and 8-1) can be identified using A549cells and gymnotic delivery of the oligonucleotide. The mediumnephrotoxic compound (comp 3-1) has reduced EGFR very close to 80% ofsaline which considering the short incubation time can be considered asindicative of potential toxicity.

Therefore it is concluded that P549 cells are useful in identifyingnephrotoxic compounds using EGFR mRNA as biomarker and gymnosis fordelivery, potentially with an incubation time of more than 2 days.

Example 7 PCSK9 Targeting Oligonucleotide Effects on EGFR in A549 CellsUsing Transfection

In this example it was tested whether a cell line that is suitable fortransfection could be applied in the method of predictingnephrotoxicity.

A549 were cultivated as described in the “Materials and methods”section. At after at 50-60% confluence the oligonucleotides weretransfected into the cells using 0.5 μl Lipofectamin (Invitrogen,11668-019) in 25 μl OptiMEM (GIBCO 31985062) and 1 ng oligonucleotide inthe same volume OptiMEM. The cells were incubated over night at 37° C.The medium was changed using A459 regular medium+10 ng/ml EGF. 48 hoursafter transfection the cells were harvested and the EGFR mRNA level,PCSK9 mRNA levels and ATP levels were measured.

The results are shown in table 8 below, and represent the average ofthree identical treatments.

TABLE 8 EGFR, ATP and PCSK9 mRNA levels from A549 cells 48 hours aftertransfection with oligonucleotide In vivo EGFR PCSK9 ATP grade of CompConc % % % toxicity # ng saline SD saline SD saline SD saline 0 100 12.9100 13.4 100 12.7 Innocuous 1-1 1 110 12.7 107 4.8 92 8.8 Medium 3-1 175 9.1 21 1.8 48 3.8 High 4-1 1 62 6.4 31 8.3 34 10.6 High 8-1 1 82 5.1153 7.4 36 20.2

These data show that EGFR can also be used as a biomarker fornephrotoxicity in transfected epithelial cells. The nephrotoxicoligonucleotides (comp 3-1 and 4-1) were predicted as being nephrotoxicat only 1 ng of oligonucleotide after just 48 hours incubation. The ATPbiomarker supports these data nicely.

The data also show that PCSK9 was knocked down by the oligonucleotidestargeting PCSK9 (comp 3-1 and 4-1), and not by the non-targetingcompounds 1-1 and 8-1.

Example 8 PCSK9 and MYD88 Targeting Oligonucleotide Effects on EGFR inA549 Cells Using Transfection

In this example it was tested whether a cell line that is suitable fortransfection could be applied in the method of predictingnephrotoxicity.

A549 were cultivated as described in the “Materials and methods”section. At after at 50-60% confluence the oligonucleotides weretransfected into the cells using 0.5 μl Lipofectamin (Invitrogen,11668-019) in 25 μl OptiMEM (GIBCO 31985062) and 1 ng oligonucleotide inthe same volume OptiMEM. The oligonucleotides comp 1-1 and 2-1 wastransfected using 10 ng. The cells were incubated over night at 37° C.The medium was changed using A459 regular medium+10 ng/ml EGF. 48 hoursafter transfection the cells were harvested and the EGFR mRNA level wasmeasured.

The results are shown in table 9 below, and represent the average ofthree identical treatments.

TABLE 9 EGFR mRNA levels from A549 cells 48 hours after transfectionwith oligonucleotide In vivo grade of Conc EGFR KIM-1 toxicity Comp # ng% saline SD % saline SD saline 0 100 0.3 100 4.3 Innocuous  1-1 10 10911.6 90 6.0 low  2-1 10 100 6.9 68 1.6 Innocuous  6-1 1 106 8.7 77 12.0High  4-1 1 71 3.9 84 12.0 High  8-1 1 65 3.6 84 4.5 Low/medium  9-1 166 4.2 91 8.0 Medium 10-1 1 73 5.3 78 3.3 Medium 11-1 1 63 2.3 90 2.9High 12-1 1 124 4.6 92 2.6 Medium 13-1 1 120 4.3 87 7.6

Of the oligonucleotides tested in this example the EGFR biomarkeridentified 5 out of 7 nephrotoxic compounds (comp 4-1, 8-1, 9-1, 10-1and 11-1). As in Example 3 comp 12-1 and 13-1 was not predicted asnephrotoxic using the EGFR biomarker or the KIM-1 biomarker.

The EGFR biomarker appears to be a good biomarker in a transfectionbased assay which has the advantage that short incubation times andsmall amounts of oligonucleotide is needed.

Example 9 Effect on ATP as Biomarker when EGFR is Blocked

In example 7 it was shown that in CACO2 cells ATP was not predictable asbiomarker. CACO2 cell proliferation and survival do not dependent on EGFin the culture conditions as described here. The present example set outto identify whether blocking EGFR on the cell line used for thenephrotoxicity assay of the invention affects the ATP biomarker.

Erlotinib, a small molecule inhibitor of EGFR kinase activity was usedin the present assay at 5 μM. Intracellular ATP levels were measured inconfluent PTEC-TERT1 after 9 days exposure to 100 μM oligonucleotidealong with 10 ng/ml of EGF in the medium with and without erlotinib. Theresults are shown in table 10, and represent the average of threeidentical treatments.

TABLE 10 Effect on intracellular ATP of oligonucleotides in the presenceof erlotinib No erlotinib 5 μM erlotinib In vivo grade ATP ATP oftoxicity Comp# % Saline SD % Saline SD Innocuous 1-1 108.61 5.43 95.543.02 low 2-1 107.67 2.93 84.31 1.07 Innocuous 6-1 102.45 2.36 104.162.82 Medium 3-1 87.67 2.19 89.59 4.74 High 5-1 82.90 3.40 89.58 4.61High 4-1 65.71 3.20 94.44 4.74 High 8-1 50.28 0.20 97.47 2.46

These data show that the ATP profile of innocuous and toxicoligonucleotides was undistinguishable in cells where EGFR wasinactivated using erlotinib. This is a strong indication that EGFRexpression is need to be functional in cells when assessing thenephrotoxicity profile of oligonucleotides in cell based assays.

Example 10 Investigation of EGF Concentrations in the Medium when UsingATP as Biomarker

As shown in example 9 it is likely that a functional EGF receptor isneeded on the cells when ATP is used as biomarker. The present exampleevaluates the effect of the EGF concentration in the culture medium onthe intracellular ATP levels as well as on the target knockdown by theoligonucleotides.

Intracellular ATP level was measured in confluent PTEC-TERT1 after 9days exposure to 100 μM oligonucleotide along with 0, 1, 3, 10, 30 and100 ng/ml of EGF in the medium. The results are shown in table 11, andrepresent the average of three identical treatments.

TABLE 11 Intracellular ATP as % of saline with varying levels of EGF inthe medium EGF ng/ml 0 1 3 10 30 90 Comp # ATP SD ATP SD ATP SD ATP SDATP SD ATP SD saline 100 4 100 5 100 6 100 4 100 4 100 11 2-1 85 3 89 6104 3 92 6 86 2 78 3 3-1 101 1 106 3 101 5 63 2 59 0 56 2 4-1 88 3 95 362 2 29 1 30 1 30 1

These results show that when applying oligonucleotide to PTEC-TERT1cells the effect on the intracellular ATP levels is dependent on the EGFconcentration in the medium, whereas the target knockdown is unaffectedby the EGF levels in the medium. At 10 ng/ml of EGF in the medium thetoxicity of the oligonucleotide compounds can be differentiated nicely,whereas below 3 ng/ml no significant differentiation can be observed.The presence of EGF in the medium is not necessary for assessing thebiomarkers EGFR (see example 7).

Example 11: Oligonucleotide Effects on EGFR mRNA in PTEC-TERT1 Cells

Compound 20-1, is also known as ISIS, 388626 is an antisenseoligonucleotide comprising 2′-O-methoxyethyl-RNA (MOE) modifications,targeting sodium-glucose co-transporter 2 (SGLT2). This compound hasbeen shown to cause reversible in vivo nephrotoxicity in humans whendosed weekly at 50 mg, 100 mg or 200 mg in a 13 week study (Meer et al2016 J Pharmacol Exp Ther Vol 359 pp280-289).

In the present example it was investigated whether this nephrotoxicitycould be predicted by measuring EGFR mRNA upon administration ofcompound 20-1 to PTEC-TERT1 cells.

The primary immortalized PTEC-TERT-1 cell line was cultured according tothe conditions described in the “Materials and methods” section. Atconfluence the cells were treated with oligonucleotide as described inthe “Materials and methods” section, in concentrations of 30, 100, 300,400 or 500 μM.

The EGFR mRNA was measured using the assays described in the “Materialsand methods” section.

The results are shown in tables 12 below and represent the average ofthree identical treatments.

TABLE 12 EGFR mRNA 9 days after oligonucleotide treatment of PTEC-TERT1cells Day 9 EGFR Conc μM % saline SD saline 100 12  30 90 16 100 72 8300 61 5 400 59 7 500 60 13

From these data it can be seen that the in vivo nephrotoxicity of theMOE antisense oligonucleotide (compound 20-1) could be predicted usingPTEC-TERT1 cells with EGFR mRNA as biomarker at oligonucleotideconcentrations above 100 μM.

1. An in vitro method for predicting in vivo nephrotoxicity of a nucleicacid molecule in a mammal, said method comprising the steps of: a.culturing cells expressing epidermal growth factor receptor (EGFR) in asuitable cell culture media; b. administering the nucleic acid moleculeto said cell culture; c. incubating the cells for a period of time; andd. subsequently measuring the EGFR mRNA level in the cells; wherein adecrease in EGFR mRNA levels is indicative of a nucleic acid moleculewhich is, or is predicted to be, associated with nephrotoxicity.
 2. Themethod according to claim 1, wherein EGFR mRNA level is compared to areference value obtained from cells treated with vehicle control or anon-toxic reference nucleic acid molecule, where the non-toxic referencenucleic acid molecule has been validated as non-toxic in vivo.
 3. Themethod according to claim 2, wherein the non-toxic reference nucleicacid molecule is an antisense oligonucleotide compound consisting ofCGTcagtatgcgAATc (SEQ ID NO: 1), wherein lower case letters representDNA units, bold upper case letters represent beta-D-oxy-LNA units, allLNA C are 5′methyl C and all internucleoside linkages arephosphorothioate linkage.
 4. The method according to claim 3, wherein alevel of EGFR mRNA below 80% relative to the vehicle control ornon-toxic reference value is predicative of nephrotoxicity of thenucleic acid molecule.
 5. The method according to claim 2 wherein EGFRmRNA level is further compared to a second reference value obtained fromcells treated with a nephrotoxic reference nucleic acid molecule, wherethe nephrotoxic reference nucleic acid molecule has been validated tocause nephrotoxicity in vivo.
 6. The method according to claim 5,wherein the toxic reference nucleic acid molecule is an antisenseoligonucleotide compound consisting of GCtgtgtgagcttGG (SEQ ID NO: 4),wherein lower case letters represent DNA units, bold upper case lettersrepresent beta-D-oxy-LNA units, all LNA C are 5′methyl C and allinternucleoside linkages are phosphorothioate linkage.
 7. The methodaccording to claim 1, wherein step d) further comprises the measurementof extracellular kidney injury molecule-1 (KIM-1) protein orintracellular mRNA levels, wherein an increase in KIM-1 levels areindicative of a nucleic acid molecule which is, or is predicted to be,associated with nephrotoxicity.
 8. The method according to claim 7,wherein a level of KIM-1 protein above 200% relative to the saline ornon-toxic reference value is predicative of nephrotoxicity of thenucleic acid molecule.
 9. The method according to claim 7, wherein alevel of KIM-1 mRNA above 1000% relative to the saline or non-toxicreference value is predicative of nephrotoxicity of the nucleic acidmolecule
 10. The method according to claim 7, wherein the increase inKIM-1 is predicative of nephrotoxicity for a nucleic acid molecule evenif the EGFR mRNA level is not decreased.
 11. The method according toclaim 1 wherein the culture media in step a) comprises at least 4 ng/mlof epidermal growth factor (EGF) and step d) further comprises themeasurement of intracellular adenosine triphosphate (ATP) levels;wherein a decrease in intracellular ATP levels is indicative of a drugsubstance which is, or is predicted to be, associated withnephrotoxicity.
 12. The method according to claim 11, wherein a level ofintracellular ATP below 80% relative to the saline or non-toxicreference value is predicative of nephrotoxicity of the drug substance.13. The method according to claim 1, wherein the cells expressing EGFRis selected from the group consisting of epithelial cell, endothelialcell and mesenchymal cells, and neuroectodermal cells.
 14. The methodaccording to claim 13, wherein the cell culture is a primary kidneyepithelial cell culture selected from the group consisting of proximaltubule epithelial cells, distal tubule epithelial cells and collectingduct epithelial cells, in particular primary human PTEC or rat PTECcells.
 15. The method according to claim 13, wherein the cellsexpressing EGFR are cultured from an immortalized cell line, such as ofhuman PTEC-TERT-1, ciPTEC, CACO2, HK-2, NKi-2 or human A549 cell lines.16. The method according to claim 1, wherein the period of incubationwith the nucleic acid molecule is between 2 and 6 days, such as around 3days.
 17. The method according to claim 1, wherein the nucleic acidmolecule is selected from a RNAi agent, an antisense oligonucleotide oran aptamer.
 18. The method according to claim 1, wherein the nucleicacid molecule comprises one or more 2′ sugar modified nucleosides,independently selected from the group consisting of 2′-O-alkyl-RNA,2′-O-methyl-RNA, 2′-alkoxy-RNA, 2′-O-methoxyethyl-RNA, 2′-amino-DNA,2′-fluoro-DNA, arabino nucleic acid (ANA), 2′-fluoro-ANA and LNAnucleosides.
 19. (canceled)
 20. The method according to claim 1, whereinthe nucleic acid molecule is an antisense oligonucleotide capable ofrecruiting RNase H.
 21. A method for selecting one or more nucleic acidmolecules for in vivo administration to a mammal, from a library ofnucleic acid molecules, said method comprising the steps of a. obtaininga library of nucleic acid molecules; b. administering each member of thelibrary of nucleic acid molecules to a cell culture expressing epidermalgrowth factor receptor (EGFR); c. culturing the cells in vitro for aperiod of time; d. measuring the amount of intracellular EGFR mRNA, andoptionally additional biomarkers, for each nucleic acid molecule, and e.selecting one or more nucleic acid molecules wherein the % reduction inEGFR relative to a reference value is above 80%. 22.-27. (canceled)